V30 wp ribograma project[1]

RIBOGRAMA PROJECT Patents and copyrights N ยบ 200701216 (2) MCT Esp. 02/18/2010 (SPAIN)

A. Ferreira-Alemao, MD PhD

MADRID/LISBOA 2013

INSTITUTO DE CIÊNCIAS BIOMÉDICAS E HUMANAS UNIVERSIDAD COMPLUTENSE DE MADRID

RIBOGRAMA PROJECT Patents and copyrights N º 200701216 (2) MCT Esp. 02/18/2010 (SPAIN)

A. Ferreira-Alemao, MD PhD

MADRID/LISBOA 2013

A. Ferreira-Alemao, MD PhD UNIVERSIDAD COMPLUTENSE DE MADRID Facultad de Medicina Madrid

ribograma@gmail.com www.ribograma.com + 351 93 671 54 88

The true joy of life is to be used for a purpose that recognize as great, be a force of nature, instead of being a measly handful of woes and grievances complaining that the world do not want to spend to make him happy.

(George Bernard Shaw in “Courage to evolve�)

RIBOGRAMA PROJECT PRESENTATION We intend to present a research and health project on cancer (RIBOGRAMA PROJECT), for an innovative method of early diagnosis of colorectal cancer (and cancers of other organs and systems), which was developed within the studies of a PhD thesis at the University Complutense of Madrid. The scope and depth of this new method of research on cancer (RIBOGRAMA) is exposed in a brief monograph on the concept and applications, which is part of this presentation and is an instrument of dissemination and promotional marketing of potentialities of this method for monitoring the changes of the phenotype of eukaryotic cells under the stimuli of the environment where they are. For example, to speak of the great technical and economic potential of this new method of investigation and monitoring of cancer of colon and rectum, we take the sample of the whole population of people living in Europe, corresponding to European Union countries, representing almost a 500 million people.

The sporadic colorectal cancer is a disease which begins substantially at about the 50 years of age. In statistical terms, the population of Europe with over 50 years of age has an expression roughly 50% of its total.

Thus, broadly speaking, about half the European population will be older than 50 years, which therefore correspond to a target/universe to study (the colorectal cancer) corresponding to 250 million. If in these people there is an adherence to the RIBOGRAMA test execution at about 10% of total value, given its great simplicity and strong powers of persuasion and information about the disease of colorectal cancer, then we have a scenario testing around 25 million people, which is carried out every 6 months, will involve the execution of 50 million tests per year!!.

According to Eurostat, annually die in the European Community, more than 200 000 people of colorectal cancer. Given these figures and given the great simplicity of implementation and interpretation of test results RIBOGRAMA, implying an early preventative health politic of authorities of each country from the perspective of public health measures on the part of the governments, it will not be difficult to imagine how easy it will reduce the actual number of deaths at least to half of those referred 200,000 deaths. This is the design of this RIBOGRAMA PROJECT. Beyond the European continent, we add the same reasoning for calculating other continents and large countries, it is easy to conclude that we are facing an exponential growth in business, and that without putting the scenario if the RIBOGRAMA method is also used in the study of cancer regarding other organs and body systems, which would add to other areas in Life Sciences and Biosciences.

The beginning and development of a project with such dimensions is only possible with a project with financial support and scientific coordination by the author and owner of the intellectual authorship of the proposal putted in his PhD thesis (at Universidad Complutense de Madrid) whose idea, already patented, will be of great utility, and may have an economic and financial returns within set between 3 and 5 years, with a clear advantage for the States, to being able to achieve in a few more years, an amount that will exceed two thousand five hundred million Euros/year, given the savings in loss of life and cost savings for the Ministry of Health and the Ministry of Labour and Social Solidarity, given the savings in costs and gains in profits derived, directly or indirectly resulting from cancers of other organs and systems, which would also be the target of the method RIBOGRAMA, both in early diagnosis, either in the direction of anti-cancer treatment.

Briefly, it can be said that we have a method eminently practical and of great economic and social impact. For the patient and the doctor, the method allows to see the cancer of colon and rectum as a chronic disease that can be controlled, as in diseases such as diabetes, dyslipidemia, in a large scale without using invasive blindly I

methodologies, unless the patient reaches the doctor with manifest symptoms of colorectal cancer. In this methodology there are practical and objective forces which allow placing patients with high curves of RIBOGRAMA in risk groups, which then have a formal indication to be studied and treated by endoscopic.

After all, there are "papers" published that speak in sharp decline, or disappearance of polyps with medical treatment on the basis of a diet and prescription of medication already licensed for cardiovascular protection and anti-inflammatory activity. With this method it is possible to significantly lower the number of deaths from colorectal cancer (and other cancers) in the countries, if we adopt sanitary measures for screening and diagnosis with the RIBOGRAMA METHOD, because the extent of populations to follow the RIBOGRAMA METHOD will be quite high.

In the case of colorectal cancer the extent of adhesion to the method will be good given the following sum of twelve "friendly" factors: 01 - No invasive act; 02 - There is no necessity for the patient to handle its own feces during the harvest; 03 - No fecal odor annoyance since the collector of feces is designed industrially accordingly; 04 - No bowel preparation is necessary; 05 - Enemas are not needed cleaning; 06 – It does not require the use of cathartics; 07 – It is not necessary to shift the patient to a medical center for a medical examination; 08 - The patient does not have to change his way of life; 09 - The patient does not have to change the routine of their daily activities or work; 10 - The patient does not need to change the pace and type of food; 11 - The patient does not have to stop, or modify any medication; 12 - No false negative or false positive results, because for the test specification merely count the ribosomes of colonocytes, which are cells that are malignized in cases of colorectal cancer, or in cases of malignant process.

The advantages of the RIBOGRAMA METHOD over other colon and rectum noninvasive cancer tests are twelve evident factors that by themselves provide a good patient compliance and preference, determinant for health professionals, because there is no false positive or false negative results, since RIBOGRAMA is considering the loads of ribosomes of desquamated cells of colorectal mucosa, which are mixed with stools, not being necessary the colonoscopy to know whether the patient is, or not, in a process of gut malignization.

The project consists of performing a proof of concept during two years, allowing the validation of results with which it is possible to make comparative studies and may be possible to build good and credible values quantifying the levels of free ribosomes, which are responsible for the production of proteins that serve to double the protein content of cells, which, in the states of malignancy do not have the universal self.

With this method it is possible to prevent people from being caught with a colorectal cancer, with invasion of the submucosa because, as can be seen, the increased amount of free ribosomes, above a certain level, defined the proof of concept in the project, to develop the RIBOGRAMA, before the occurrence of any polyp, it is easy to know that a patient began to have an important criterion to be included into a risk group, in which even if there is no character of malignant lesion, at a histological level, such as a seemingly benign polyps, there is already a biochemical profile of malignancy. Within the global scientific community, this method utilizes a new way to study the biochemical behaviour of the malignization. It will impact on the economy (and will give unparalleled statistical indices) in the countries. That is, before there is any malignant lesion, visible in optical microscopy (pathologic anatomy), before a colonoscopy, it will be possible the doctor to be sure whether there is a process of cellular proliferation, translated by a noticeable and significant excess of free ribosomes in cytoplasm of colonocytes, which is a common characteristic of a malignant tissue, or with a tendency to becomes malignant.

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Against this method, the cell/tumor markers, which are proteins, are less sensitive to study the malignancy, since they are elements manufactured in the free ribosomes. It means to say that these (free ribosomes) are the main elements for monitoring the malignant phenotype (upstream) in the chain of cell production/proliferation, since the cell/tumor markers are downstream to the variation curve of concentration of the free ribosomes (RIBOGRAMA curve). The method (RIBOGRAMA) allows, in a non-invasive way, with easy adhesion of the population, to measure the quantity of free ribosomes in the colonocytes (which are mixed in the faeces), by studying the pattern of them in the colorectal mucosal cells, being made possible harvest from collecting the stool in the toilet-fit of the bathroom, in the house of each patient that chooses to conduct the test, without to manipulate their own feces.

As is described in the Abstract, attached to this approach, the method gives a concrete numerical result, and is convincing to the patient's household and those with whom he speaks and lives in the day-to-day, making that other people are induced to do it, as with the control of treatable and controllable diseases (diabetes, dyslipidemia, uric acid, for example). The patient just take home a "collector" of feces (whose design is explained in the RIBOGRAMA project), who will buy it in a pharmacy, a chemist, or a shopping center and, after sampling of feces, at home, guided by an illustrated instruction booklet, will deliver the biologic product in a clinical laboratory, which has its technical suitability and accreditation.

The practical consequences of this method are such that there is a marked decrease in spending resulting from the treatment of colorectal cancer, and a decrease of suffering resulting from the significantly lower number of patients with colorectal cancer, because people know when to go to a doctor's appointment, when the curve of his RIBOGRAMA provides information on the early tendency of the colorectal mucosa to have the risk to becomes malignant, which arise as a result of a significant increased and persistent high RIBOGRAMA curve before any colonoscopy. Patients may repeat the RIBOGRAMA test within a short periodicity at low cost, without the risk of being caught with a lesion on the path to malignancy, with great cost savings to the countries and to the community without having to make a blind colonoscopy, with discomfort with its preparation and its realization.

Speaking of costs with the disease, once diagnosed cancer in a stage of which exceeded the submucosa we can speak in multiple charges, as a result of colorectal cancer, which can be listed in the following list: 01 - Costs of operating theater; 02 - Charges for intensive care and/or the recovery room of patients operated from the cancer; 03 - Charges for hospitalization of the patient as a result of the operation; 04 - Charges for chemotherapy and other drugs; 05 - Charges to follow up exams; 06 - The Social Security System no longer receives input from an inactive citizen and is paying to an incapacitated; 07 - Usually the patient will not go alone to a consultation and follow up treatments, taking with him a family member or a friend who will not work in his workplace, with loss of productivity to the community.

It would be useful to present a Symposium related to the RIBOGRAMA topic, aimed at biomedical researchers, University teachers, entrepreneurs and investors on the subject, focusing on the multiple aspects related to the phenotypic behavior of a cell in the process of malignant transformation to reveal the potential of the method, which become a universal tool for study and research in oncology and other areas such as the toxicity of substances on the cellular homeostasis in the pharmaceutical and food industry.

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ESSENCE AND ADVANTAGES OF THE METHOD 01- There is well established evidence (and recent) emphatically argues that the idea that RNA content is elevated in cancer cells and that genetic events that lead to cancer are often linked directly and indirectly to the ribosome biogenesis; 02- Thus, the significant increase in the number of accumulated free ribosomes in the cytoplasm of a community of cells from any tissue (by counting with a flow cytometer, after isolated and labeled with fluorescent particles -- fluorochromes) can constitute one phenotypic expression aspects of malignancy of the fine structure of the transformed cells; 03- In cells undergoing malignant transformation, increased number of free ribosomes above a certain level of concentration (per unit volume) to be established as the limit of normalcy pattern constitutes a measurable sign of the trend of expression malignancy. The verification of this increase is seen before the global fine morphological structure of a transformed cell or tissue to which it belongs, assume a malignant character set; 04- The phenotypic expression represented by the significant increase of free ribosomes is characteristic of neoplastic growth and may represent a predictive aspect of cell proliferation rate and prognosis of the patients; 05- Morphological and biochemical studies suggests that the ribosomes of mammalian cells are free in the cytoplasmic matrix (free ribosomes), or bound to the cytoplasmic membrane as part of the rough endoplasmic reticulum (membrane bound ribosomes); 06- There morphological and physicochemical evidence that distinct tissues of poorly differentiated differ in that the latter have a higher proportion of free ribosomes; 07- The production of the ribosome is an important metabolic activity related to cell growth and recent data suggest that the nucleolus also plays an important role in regulating the cell cycle, senescence and stress responses; 08- The ribosome biogenesis involves rRNA synthesis, maturation and the “assembly" of RNA and ribosomal proteins in ribosomal subunits, small and large; 09- The ribosome biogenesis is regulated through the cell cycle, primarily at the level of rRNA synthesis; 10- Oncobiologic researchers have shown that the expression of abnormal rDNA and genes of ribosomal proteins are associated with the development of tumors as well as various cytological studies show that the nucleoli (sites where rRNA is synthesized and ribosomes are " assembled ") of the cancer cells are increased because they have an increased transcriptional activity, representing predictive aspects of the rate of cell proliferation and the prognosis of the patients, which is why, the cancer cells have a high expression of the ribosomal proteins, revealing higher ribosome content, which explains that the ribosome biogenesis and oncogenesis are intimately connected; 11- Because of its size (25 nanometers) ribosomes can only be studied with the resources of nanotechnology; 12- The present study shows that it is possible to define some guiding principles on the value and meaning of the increased number of free ribosomes above a certain concentration level established as the limit of normal; 13- This significant progressive increase in the number of free ribosomes represents a clinical manifestation of the dynamics of the biochemical process to the malignancy of the cells in a specific cell community of tissue or organ; 14- The verification in the field of electron microscopy observations showed the existence of large quantities of free ribosomes in neoplastic or pre-malignant tissues. With flow cytometry also check the same, but measurable way, after isolation, labeling with fluorescent dyes and quantification of free ribosomes, which allows comparisons; IV

15- Check of such large amounts of free ribosomes can be translated into a graph curve that represents and defines, over time, the predisposition for malignization growth of a tissue, cell or community under certain conditions; 16- This graph curve will have the designation of RIBOGRAMA. Has a (may be reproducible in equivalent circumstances) mathematical basis corresponding to a non- subjective language; 17- Analysis of trends and variations found in that curve will be a reliable indicator of the degree of evolution and propensity for malignization; 18- The RIBOGRAMA corresponds to a phenotypic appearance with quantitative, that can provide quantitative information, and risk levels of significance, prior to the "open" malignization, and profile information of hereditary familial risk and food profile, dietary and lifestyle habits; 19- The potential power of the RIBOGRAMA method is of interest to evaluate the effectiveness of a substance with antiproliferative effect on a community of cells under study and surveillance. This method also has capacities for monitoring the phenotypic behavior of the cells when certain substances appear in their environment which affect the proliferative status.

20- Indeed, because the variation in quantity of free ribosomes, an important parameter of evaluation index proliferation, it makes sense considering that the RIBOGRAMA curve reflects the nature of the response to a stimulus on a given cell, either in inhibition of proliferation, whether in stimulating their proliferation.

21- The free ribosomes are responsible by the internal economy of the cell in the production of proteins and their variation quantity (being more, or less) reflect the data interpretation of the feature how a cell is behaving in a existing agent in the medium where the cells of certain cell type community exist.

22- Compared to existing non-invasive tests in use (blood in the stool and DNA mutation analysis of coloncyts) counting test ribosome has, theoretically, some features that are major advantages over existing tests: i.

Knowing that will be studied as isolated coloncyts own and specific technique, the specificity of the results is 100% relative to the count of free ribosomes; ii.

The existence of levels amounts of free ribosomes, risk criteria and significance, allowing early preventive and therapeutic approaches to malignant tendenc, while the discovery test DNA mutations in the stool, the only answer is positive/negative, not allowing a quantification by levels or degrees with a significant risk; iii.

The test DNA mutations in stool has false positives and false negatives, which vary to a significant extent, according the authors; iv.

The positive test for occult blood in the stool has a high percentage of false positives and false negatives are not negligible; v. When a tumour bleeds, this means that the mucosal barrier has been already destroyed by erosion/invasion of the submucosa, with its blood and lymph vessels, not ensuring safety regarding metastization and nodal involvement, that is, no tumor Tis is a T0; vi.

Mutations in DNA tests on stool may be from other organs (respiratory tract, digestive tract, a pillar of colorect, etc.), Allowing no guarantee of eventual location of a tumour; vii.

In addition, mutations in these tests (DNA mutations in faeces) may be non-mutant phenotypes, which contributes to the abundant false positives; viii.

The counting test free ribosomes, corresponding to significantly increased the number of free ribosomes in colonocytes isolated from faeces levels, represents the existence of a true increase in protein synthesis uncontrolled unable, therefore, be false positive, because a tumor depends essentially on an exaggerated production of proteins in the growth process, and that the cell can only be done by free ribosomes; ix.

Potential in clinical use in monitoring and diagnosis RIBOGRAMA and "follow up" cancer colorect.

23- The hypothesis on stage RIBOGRAMA utilization as an expression of genotypic behaviour coloncyts (free ribosomes express density in units per volume over time), open the door to many fields of clinical and laboratory research. Enable tracing and early diagnosis of colorectal cancer in conditions conducive to convince the family to also undergo a noninvasive test that does not require anyone to V

manipulate their feces, and convincing, if RIBOGRAMA elevated curves, to undergo, justifiably, a diagnostic and therapeutic colonoscopy, once there are no clinical signs; 24- Through RIBOGRAMA of cells shed from the colorectal mucosa, conveyed in feces and collected in an appropriate and specific container without patient handling their feces, using a design created and suitable for this purpose, people would have a simple means for attachment to this diagnostic widespread screening, and could be oriented towards an endoscopic examination, or be routed to a risk group, and be observed and later studied graphic profiles of the numerical values of free ribosomes (RIBOGRAMA) comparatively drawn to the study of large population groups - median standard curve.

A. Ferreira-Alemao, MD PhD UNIVERSIDAD COMPLUTENSE DE MADRID Facultad de Medicina Madrid

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RIBOGRAMA PROJECT

General Index FOREWORD ........................................................................................................................... 1 I - THE RIBOGRAMA METHOD - (PROJECT TO IMPLEMENT ITS APPLICATION IN THE DAILY CLINICAL PRATICE) ........................................................................................... 2 II - NEW APROACH TO THE STUDY OF THE COLON AND RECTUM CANCER ................................................................................................ 4 1- APPROACH TO THE TOPIC ............................................................................................................. 8 2 – OVERVIEW .............................................................................................................................................. 8 3 - ESSENCE OF THE METHOD ........................................................................................................... 9 4 - SCOPE AND POTENTIAL OF THE METHOD ......................................................................... 10 5 - MOTIVATION AND ARGUMENTS FOR USING THE METHOD ...................................... 10

III - TRADITIONAL (ACTUAL) COLORECTAL CANCER SCREENING ............................................................................................................ 13 IV - STATISTICAL DATA ON COLORECTAL CANCER (CRC) ............................................................................................................................ 17 1 - FECAL OCCULT BLOOD TEST (FOBT) ................................................................................... 17 2 – SIGMOIDOSCOPY ............................................................................................................................. 20 3 - COMBINATION OF FOBT AND FLEXIBLE SIGMOIDOSCOPY ..................................... 22 4 - BARIUM ENEMA ................................................................................................................................. 23 5 - COLONOSCOPY ................................................................................................................................. 23 6 - VIRTUAL COLONOSCOPY (COMPUTED TOMOGRAPHIC [CT] COLONOGRAPHY) ........................................................................................................................... 25 7 - DETECTION OF DNA MUTATIONS IN THE STOOL ........................................................... 27

V - CRC EPIDEMIOLOGY AND RESOURCE ALLOCATION ............... 32 1 - CRC INCIDENCE AND MORTALITY .......................................................................................... 32 2 - CRC SURVIVAL ................................................................................................................................... 33

VI - CRC RESOURCE ALLOCATION IN EUROPE AND AUSTRALIA ............................................................................................................. 34 VII - CANCER AND COLORECTAL CANCER SPENDING ................... 37 VIII - RESOURCE ALLOCATION FOR SCREENING ............................... 38 i

RIBOGRAMA PROJECT IX - CRC RESOURCE ALLOCATION TO DIAGNOSIS AND TREATMENT PROGRAMMES ..................................................................... 40 X - RESUME ........................................................................................................................ 41 XI - BIOMARKERS FOR EARLY DETECTION OF COLON CANCER – THE LAST TEN YEARS ......................................................... 44 1 - Introduction ........................................................................................................................................... 44 2 - Biomarkers of Risk versus Early Detection.......................................................................... 45 3 - Biomarkers for Risk .......................................................................................................................... 45 4 - Biomarkers for Early Detection................................................................................................... 46 5 - APC ............................................................................................................................................................ 47 6 - MMR .......................................................................................................................................................... 47 7 - K-ras.......................................................................................................................................................... 47 8 - p53 ............................................................................................................................................................. 48 9 - Kinases .................................................................................................................................................... 48 10 - Serum Markers .................................................................................................................................. 49 11 - L-DNA..................................................................................................................................................... 49 12 - β-Catenin .............................................................................................................................................. 49 13 - bcl-2 Gene ........................................................................................................................................... 50 14 - Proliferation in Aberrant Crypt Foci ....................................................................................... 50 15 - Cyclin D1 .............................................................................................................................................. 50 16 - Cyclin E1 .............................................................................................................................................. 50 17 - SMAD4 ................................................................................................................................................... 51 18 - Panel of Markers .............................................................................................................................. 51 19 - Biomarkers as Intermediate End Points in Colorectal Cancer Prevention ............................................................................................................................................ 51 20 - Adenomas as a Logical Surrogate End Point ................................................................... 52 21 - Nonsteroidal Chemopreventive Agents and Biomarkers ........................................... 52

XII - POTENTIAL OF THE RIBOGRAMA PROJECT ................................ 59 XIII - INTERPRETATION OF THE RIBOGRAMA CURVE .................... 63 XIV - RIBOGRAMA – IS A BIOSENSOR TO MONITOR CARCINOGENESIS ............................................................................................. 65 XV - RIBOGRAMA - A NEW METHOD IN THE STUDY OF CARCINOGENESIS – (BIOMOLECULAR CONCEPT AND ITS USE IN CLINICAL ONCOLOGY AND LABORATORY ANALYZE) ............................................................................ 66 ii

RIBOGRAMA PROJECT XVI – RIBOGRAMA: CONCEPT AND CLINICAL APPLICATIONS IN CANCER THERAPY - (A NEW METHOD TO SURVEY CARCINOGENESIS SCREENING, DIAGNOSIS AND THERAPEUTIC) .......................... 68 1 - ABSTRACT ............................................................................................................................................ 68 2 – INTROIT .................................................................................................................................................. 68 3 - A PERSPECTIVE ON THE BIOLOGY OF MALIGNANT CELLS .................................... 69 4 - ACCUMULATION OF RIBOSOMES IN THE PROCESS OF ONCOGENESIS ......... 70 5 - POTENTIAL BIOMEDICAL APPLICATIONS RESULTING FROM THE COUNT OF RIBOSOMES ............................................................................................................... 70 6 - THE NUCLEOLUS............................................................................................................................... 71

XVII - INTRODUCTION TO THE STUDY OF THE VERY EARLY DIAGNOSIS (PRE-IN SITU STAGE) OF COLORECTAL CANCER (CRC) .................................................................. 73 XVIII - PROCEDURES FOR PREPARATION AND COUNTING FREE RIBOSOMES (TECHNICAL PHASES FOR CONCRETIZATION OF THE RIBOGRAMA METHOD) .................................................................................. 76 1 - THE ESSENCE OF THE METHOD .............................................................................................. 76 2 - BIOMOLECULAR BASE OF THE CELLULAR MACHINERY .......................................... 81 3 - RIBOGRAMA versus TUMOR MARKERS IN THE DIAGNOSIS AND MONITORING ACCURACY IN CLINICAL PRACTICE ...................................................... 81 4 - PURPOSE AND POTENTIAL OF THE METHOD .................................................................. 83 5 - MOTIVATION AND RATIONALE FOR THE METHOD........................................................ 83 6 - EXECUTION AND STEPS TO ACCOMPLISH THE METHOD ......................................... 85 7 - QUANTITATIVE PCR: TECHNICAL ADDENDUM ................................................................ 89

XIX - CAPSULE ................................................................................................................. 91 XX - NUEVOS CAMINOS EN LA INVESTIGACIÓN DEL CANCER ..................................................................................................................... 92 XXI - METFORMIN PREVENTS CARCINOGEN - INDUCED TUMORIGENESIS - (METFORMIN PREVENTS CANCER) ................................................................................................................. 142 XXII - SIMVASTATIN INHIBITS CANCER CELL GROWTH ............. 150

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RIBOGRAMA PROJECT XXIII - NONSTEROIDAL ANTI-INFLAMMATORY DRUGS AS ANTICANCER AGENTS: MECHANISTIC, PHARMACOLOGIC, AND CLINICAL ISSUES ................................ 162 XXIV - EXTRACOLONIC TUMOURS IN PATIENTS WITH LYNCH II SYNDROME ................................................................................... 184 XXV - CLINICAL AND LABORATORY APPLICATIONS OF AMNIOTIC MEMBRANE AND FLUID IN STEM CELL BIOLOGY ................................................................................................................. 199 XXVI - PRESERVATION SOLUTION FOR COLONOCYTES ............ 218 XXVII - SUPPORT PROTOTYPE OF FAECES COLLECTION (COLLECTOR) .................................................................... 222 XXVIII – TREATMENT COSTS OF BOWEL CANCER IN ENGLAND................................................................................................................ 230 XXIX - ECONOMIC AND FINANCIAL ANALYSIS OF ENGLISH PRESS FOR THE CRC ............................................................ 236 XXX - MEDICATIONS WITH ANTIPROLIFERATIVE PROPRIETIES ...................................................................................................... 240 XXXI - APTAMERS ....................................................................................................... 241 XXXII - CANCER-SELECTIVE ANTIPROLIFERATIVE ACTIVITY IS A GENERAL PROPERTY OF SOME GRICH OLIGODEOXYNUCLEOTIDES ..................................................... 246 XXXIII – PATENTES AND COPYRIGHT Nº 200701216 (2) MCT ESP. 02/18/2010 (SPAIN) ................................................................ 258

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RIBOGRAMA PROJECT

FOREWORD Molecular Biology techniques allowed a dramatic change in the paradigm of human disease in the last decade.

Before the Era of Genomics, diseases with a genetic background were only considered from a qualitative point of view; a mutation in a gene may lead to a mutation of the corresponding protein with phenotypic consequences. Nowadays, modern medicine relies on the quantitative paradigm of the disease, in which the levels of genomic output quantified as RNA or proteins are essential variables of the whole disease landscape.

Genomic output is directly related with cell function, and in disease this output can be related with phenotype but also with the susceptibility and response to medical treatments. In consequence, quantitative knowledge of the genomic output is an essential factor in modern medicine.

In the last twenty years the medical community has observed, analyzed and applied many of the molecular biology techniques for the analysis of genomic contents and activity. From the Sanger’s sequence technique to the new platforms for next generation sequencing, many diseases have been analyzed from the point of view of the Genetics, with the main objectives of searching for biomarkers of diagnosis, prognosis and treatment targets. However, genetic biomarkers are often not easy to detect and analyze alone in the context of the disease progress.

Cancer is one of the best examples of the advances of quantitative medicine. In fact, data obtained from the quantitative analysis of genomic and proteomic output in diverse cancers have allowed to design more efficient anti-cancer drugs, and to convert a deadly disease into a chronic one. Indeed, because of the intrinsic multifactorial nature of cancer, reliable biomarkers are still a matter of investigation for tumors. The complex nature of tumorigenesis made this process difficult to analyze because it often shows entangled gene expression patterns.

Cancer cells are characterized by an accelerated metabolism that facilitates their division and progression, including also other characteristics such as the contact inhibition. The extreme metabolic needs of tumor cells could be characterized by the measurement of “vital” parameters. The RIBOGRAMA project is a molecular platform based on the field of quantitative genomics, with the main objective of cancer diagnosis and prognosis. RIBOGRAMA platform will take advantage of the use of ribosomes as biomarkers for cancer diagnosis and stratification, open a new field in molecular diagnosis.

Francisco J. Enguita, PhD INSTITUTE OF MOLECULAR MEDICINE FACULTY OF MEDICINE Cellular Biology Unit, 3rd Floor University of Lisbon Ramil Ramazanov, MD PhD INSTITUTO DE CIENCIAS BIOMÉDICAS E HUMANAS Lisbon A. Ferreira-Alemao, MD PhD UNIVERSIDAD COMPLUTENSE DE MADRID Facultad de Medicina Madrid 1

RIBOGRAMA PROJECT

I - THE RIBOGRAMA METHOD -

(PROJECT TO

IMPLEMENT ITS APPLICATION IN THE DAILY CLINICAL PRATICE) Disease prevention is receiving more attention in the Western type food and life. This welcome trend is largely a result of two economic forces: The increased level of support for prevention research by the Health Ministery of the countries (Healthy People 2000), and the potential payoff of prevention research in cutting the health care costs of managing patients with end-stage diseases. Indeed, the efficacy of preventive measures for many chronic diseases (such as heart disease and cancer) are being evaluated with the full expectation that mortality and morbidity — and perhaps health care costs — will decrease.

One example of recent cancer prevention research indicates that there may be a 40–50% reduction in mortality from colorectal cancer in persons who take aspirin or other nonsteroidal antiinflammatory drugs (NSAIDs) on a regular basis. It is clear that an effect of this magnitude would have a significant impact on both the number of lives saved and on the health care dollars recovered. Colorectal cancer is a major cause of death in Western civilizations, claiming about 55,000 lives in the United States on the end of last century.

People with life of Western type have a 1 in 20 lifetime risk of developing this disease, and 1 in 10 have a family member who develops colorectal cancer. Initially, it was hoped that early detection would reduce these high numbers. However, despite recent evidence that early detection by fecal occult blood tests and flexible sigmoidoscopy can decrease the risk of colorectal cancer death by 20–30%, most people do not undergo appropriate screening.

Therefore, a number of laboratories have initiated research efforts focused on understanding the molecular basis of the potential chemoprotective effects resulting from aspirin and other NSAID use. It is hoped that this approach, in conjunction with more rigorous clinical trials, will lead to a rationale design for future colorectal cancer prevention regimens. This perspective considers both the clinical and the basic research efforts underway presently, around 100 years of the discovery of aspirin.

THE RIBOGRAMA METHOD The option to foresee the strategy of this work lies in the fact that the concept and applications of the method RIBOGRAMA be extended to various fields of technical-scientific activities in the field of Life Sciences, among which stands out the medicine designed to humans, in addition to others, such as Public Health, Food Industry, Pharmaceutical, Veterinary Medicine Research in Clinical Trials, Research Oncology, Clinical Research and Laboratory, medical and Hospital Practice, etc., which fits into INSTRUMENT OF CREATION FOR MORE AND BETTER JOBS in the field of innovation and development, towards reducing the differences in prosperity and living standards in EU regions, promoting thereby the economic and social cohesion.

This research work was carried out following the development of an idea, in a Doctoral program at the University Complutense of Madrid, which relates to the quantification of biochemical dynamics of the biocelular process of proliferation, as a practical aspect of the process of cell proliferation and the main phenomena visible connected to the oncogenesis. In the development of cytological structure enters 2

RIBOGRAMA PROJECT a new concept in the way how we can look the permanent change in the lives of a community of cells that can only behave in three ways, in the implications of its destination: - Increased proliferation (cellular proliferation); - Involution cytological (cell apoptosis) - Maintaining the status cell.

To date these three stages are able to describe only a morphological form (microscopically speaking) method that is a subjective vision in how each investigator/technician sees the semantics of the morphology of a tissue when he looks through the microscope the cytological/histological study of the image. This was precisely the reason for the beginning of this investigation that led to an exhausting work (with 1174 retrospective research references), which as a whole has been proposed as a PhD thesis before a Scholar Court, at the University Complutense of Madrid, which assigned the maximum rating, unanimously, with honors, and put into the public debate. With the completion of this academic work emerged, so, the concept of RIBOGRAMA, (word whose etymology results from the junction and contraction of two other words ribosome and ...gram/chart), which presents the following idea:

ABSTRACT “RIBOGRAMA is a method of diagnosis, screening and monitoring the colon and rectum cancer founded on technical and current concepts of Molecular Biology. It allows monitoring the behaviour of the eukaryotic cells, in a stage previous to a "pre-carcinoma in situ” diagnosis, since it is based in the phenotype of mucosal cells composed of very high quantities of free ribosomes in its cytoplasm, which can be quantified by means of the of the flow cytometry, after its isolation, and labelled with fluorochromes. The repeated records of those quantities of free ribosomes establish a graphic curve which, above a certain level of concentration, represents a malignant tendency. It allows to say that the cells in any tissue (e.g. of the colon and rectum) can be considered in a process of developing carcinoma in the colon-rectum mucosa before any macroscopic viewing (endoscopy). This means, we are facing a new method, on a clear biomolecular level.

Thanks to this method, it is possible to have a screening program (through a non-invasive method with great acceptance by the population), in which all citizens who present a great curve of RIBOGRAMA must be checked with a mandatory therapeutic and diagnostic endoscopy, at the same time, remain under observation and therapy (food diet survey and non-steroidal anti-inflammatory and acetylsalicylic acid), with proven efficiency in the prevention and reduction of the formation of polyps or premalignant tendency injury, representing savings in the therapeutic inversion of cancer.”

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II - NEW APROACH TO THE STUDY OF THE COLON AND RECTUM CANCER We intend to present a research and health project on cancer (RIBOGRAMA PROJECT), for an innovative method of early diagnosis of colorectal cancer (and cancers of other organs and systems), which was developed within the studies of a PhD thesis at the University Complutense of Madrid. The scope and depth of this new method of research on cancer (RIBOGRAMA) is exposed in a brief monograph on the concept and applications, which is part of this presentation and is an instrument of dissemination and promotional marketing of potentialities of this method for monitoring the changes of the phenotype of eukaryotic cells under the stimuli of the environment where they are. For example, to speak of the great technical and economic potential of this new method of investigation and monitoring of cancer of colon and rectum, we take the sample of the whole population of people living in Europe, corresponding to European Union countries, representing almost a 500 million people.

The sporadic colorectal cancer is a disease, which begins substantially at about the 50 years of age. In statistical terms, the population of Europe with over 50 years of age has an expression roughly 50% of its total.

Thus, broadly speaking, about half the European population will be older than 50 years, which therefore correspond to a target/universe to study (the colorectal cancer) corresponding to 250 million. If in these people there is an adherence to the RIBOGRAMA test execution at about 10% of total value, given its great simplicity and strong powers of persuasion and information about the disease of colorectal cancer, then we have a scenario testing around 25 million people, which is carried out every 6 months, will involve the execution of 50 million tests per year!!

According to Eurostat, annually die in the European Community, more than 200.000 people of colorectal cancer. Given these figures and given the great simplicity of implementation and interpretation of test results RIBOGRAMA, implying an early preventative health politic of authorities of each country from the perspective of public health measures on the part of the governments, it will not be difficult to imagine how easy it will reduce the actual number of deaths at least to half of those referred 200.000 deaths. This is the design of this RIBOGRAMA PROJECT. Beyond the European continent, we add the same reasoning for calculating other continents and large countries, it is easy to conclude that we are facing an exponential growth in business, and that without putting the scenario if the RIBOGRAMA method is also used in the study of cancer regarding other organs and body systems, which would add to other areas in Life Sciences and Biosciences.

The beginning and development of a project with such dimensions is only possible with a project with financial support and scientific coordination by the author and owner of the intellectual authorship of the proposal putted in his PhD thesis (at Universidad Complutense de Madrid) whose idea, already patented, will be of great utility, and may have an economic and financial returns within set between 3 and 5 years, with a clear advantage for the States, to being able to achieve in a few more years, an amount that will exceed two thousand five hundred million Euros/year, given the savings in loss of life and cost savings for the Ministry of Health and the Ministry of Labour and Social Solidarity, given the savings in costs and gains in profits derived, directly or indirectly resulting from cancers of other organs and systems, which would also be the target of the method RIBOGRAMA, both in early diagnosis, either in the direction of anti-cancer treatment.

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RIBOGRAMA PROJECT Briefly, it can be said that we have a method eminently practical and of great economic and social impact. For the patient and the doctor, the method allows to see the cancer of colon and rectum as a chronic disease that can be controlled, as in diseases such as diabetes, dyslipidemia, in a large scale without using invasive blindly methodologies, unless the patient reaches the doctor with manifest symptoms of colorectal cancer.

In this methodology there are practical and objective forces which allow placing patients with high curves of RIBOGRAMA in risk groups, which then have a formal indication to be studied and treated by endoscopic.

After all, there are "papers" published that speak in sharp decline, or disappearance of polyps with medical treatment on the basis of a diet and prescription of medication already licensed for cardiovascular protection and anti-inflammatory activity. With this method it is possible to significantly lower the number of deaths from colorectal cancer (and other cancers) in the countries, if we adopt sanitary measures for screening and diagnosis with the RIBOGRAMA METHOD, because the extent of populations to follow the new METHOD will be quite high.

In the case of colorectal cancer the extent of adhesion to the method will be good given the following sum of twelve "friendly" factors: 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

No invasive act; There is no necessity for the patient to handle its own feces during the harvest; No fecal odor annoyance since the collector of feces is designed industrially accordingly; No bowel preparation is necessary; Enemas are not needed cleaning; It does not require the use of cathartics; It is not necessary to shift the patient to a medical center for a medical examination; The patient does not have to change his way of life; The patient does not have to change the routine of their daily activities or work; The patient does not need to change the pace and type of food; The patient does not have to stop, or modify any medication; No false negative or false positive results, because for the test specification merely count the ribosomes of colonocytes, which are cells that are malignized in cases of colorectal cancer, or in cases of malignant process.

The advantages of the RIBOGRAMA METHOD over other colon and rectum noninvasive cancer tests are twelve evident factors that by themselves provide a good patient compliance and preference, determinant for health professionals, because there is no false positive or false negative results, since RIBOGRAMA is considering the loads of ribosomes of desquamated cells of colorectal mucosa, which are mixed with stools, not being necessary the colonoscopy to know whether the patient is, or not, in a process of gut malignization.

The project consists of performing a proof of concept during two years, allowing the validation of results with which it is possible to make comparative studies and may be possible to build good and credible values quantifying the levels of free ribosomes, which are responsible for the production of proteins that serve to double the protein content of cells, which, in the states of malignancy do not have the universal self.

With this method it is possible to prevent people from being caught with a colorectal cancer, with invasion of the submucosa because, as can be seen, the increased amount of free ribosomes, above a certain level, defined the proof of concept in the project, to develop the RIBOGRAMA before the occurrence of any polyp, it is easy to know that a patient began to have an important criterion to be included into a risk group, in which even if there is no character of malignant lesion, at a histological level, such as a seemingly benign polyps, there is already a biochemical profile of malignancy. Within the global scientific community, this method utilizes a new way to study the biochemical behaviour of the malignization. It will impact on the economy (and will give unparalleled statistical indices) in the countries. That is, before there is any malignant lesion, visible in optical 5

RIBOGRAMA PROJECT microscopy (pathologic anatomy), before a colonoscopy, it will be possible the doctor to be sure whether there is a process of cellular proliferation, translated by a noticeable and significant excess of free ribosomes in cytoplasm of colonocytes, which is a common characteristic of a malignant tissue, or with a tendency to becomes malignant.

Against this method, the cell/tumor markers, which are proteins, are less sensitive to study the malignancy, since they are elements manufactured in the free ribosomes. It means to say that these (free ribosomes) are the main elements for monitoring the malignant phenotype (upstream) in the chain of cell production/proliferation, since the cell/tumor markers are downstream to the variation curve of concentration of the free ribosomes (RIBOGRAMA curve).

The method (RIBOGRAMA) allows, in a non-invasive way, with easy adhesion of the population, to measure the quantity of free ribosomes in the colonocytes (which are mixed in the faeces), by studying the pattern of them in the colorectal mucosal cells, being made possible harvest from collecting the stool in the toilet-fit of the bathroom, in the house of each patient that chooses to conduct the test, without to manipulate their own feces.

As is described in the Abstract, attached to this approach, the method gives a concrete numerical result, and is convincing to the patient's household and those with whom he speaks and lives in the day-to-day, making that other people are induced to do it, as with the control of treatable and controllable diseases (diabetes, dyslipidemia, uric acid, for example). The patient just take home a "collector" of feces (whose design is explained in the RIBOGRAMA project), who will buy it in a pharmacy, a chemist, or a shopping center and, after sampling of feces, at home, guided by an illustrated instruction booklet, will deliver the biologic product in a clinical laboratory, which has its technical suitability and accreditation.

The practical consequences of this method are such that there is a marked decrease in spending resulting from the treatment of colorectal cancer, and a decrease of suffering resulting from the significantly lower number of patients with colorectal cancer, because people know when to go to a doctor's appointment, when the curve of his RIBOGRAMA provides information on the early tendency of the colorectal mucosa to have the risk to becomes malignant, which arise as a result of a significant increased and persistent high RIBOGRAMA curve before any colonoscopy. Patients may repeat the RIBOGRAMA test within a short periodicity at low cost, without the risk of being caught with a lesion on the path to malignancy, with great cost savings to the countries and to the community without having to make a blind colonoscopy, with discomfort with its preparation and its realization.

Speaking of costs with the disease, once diagnosed cancer in a stage of which exceeded the submucosa we can speak in multiple charges, as a result of colorectal cancer, which can be listed in the following list: 1.

2.

3.

4.

5.

6.

Costs of operating theater; Charges for intensive care and/or the recovery room of patients operated from the cancer; Charges for hospitalization of the patient as a result of the operation; Charges for chemotherapy and other drugs; Charges to follow up exams; The Social Security System no longer receives input from an inactive citizen and is paying to an incapacitated; 7. Usually the patient will not go alone to a consultation and follow up treatments, taking with him a family member or a friend who will not work in his workplace, with loss of productivity to the community.

It would be useful to present a Symposium related to the RIBOGRAMA topic, aimed at biomedical researchers, University teachers, entrepreneurs and investors on the subject, focusing on the multiple aspects related to the phenotypic behavior of a cell in the process of malignant transformation to reveal the potential of 6

RIBOGRAMA PROJECT the method, which become a universal tool for study and research in oncology and other areas such as the toxicity of substances on the cellular homeostasis in the pharmaceutical and food industry.

(smt: - every six months)

Interpretation of RIBOGRAMA curve: → Curve that starts at the normal level, and reaching the level increased, which means care for a prophylactic study on the reasons or causes which determine its occurrence. The cells at this level (increased) still have no safety features of malignancy, but increased their number continues to grow free ribosomes, the cell is transformed into a malignant phenotype (alarm level). At this point it is essential there be a food-study survey and identification of potential mutations responsible for non-controlled increase of ribosome biogenesis in the process of carcinogenesis; → Segment of the curve derived from the anterior (segment ab), which corresponds to the phenotypic alterations and visible light microscopy, in which cells are already present with phenotypic characteristics of malignancy; → Segment of the curve, coming from the anterior segment, which already represents the level of signs and symptoms of the disease CCR (clinical level); → This segment of the curve corresponds to a change in direction of the curve ab to result from a preventive and therapeutic (medications and special diet); → Corresponds to a curve in which cells grow (multiply or proliferate) within a self-monitoring increased proliferation mechanisms, as in the case of the seminiferous tubules, or the endometrium. This increased level of ribosome biogenesis, control of these lies within the physiological mechanisms (nonmalignant); → Normal curve; → This work is intended to study item colorectal cancer (CRC), which ribograma curve is high, so it is not appropriate to talk about the meaning of it on the lower level, which still serve to study conditions of degeneration and involution of tissues and cells, whether the intestinal mucosa, or other extra-intestinal tissue.

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The increased focus on the path of Life Sciences relates to the fact that they comprise multiple fields of science that involve the scientific study of living organisms, such as plants, animals and humans, as well as bioethical considerations, with which can address the verification and the results of tests facing barriers ethical and professional conduct of the trial by a new method to study cell behavior in the face of environmental influences on eukaryotic cells, called RIBOGRAMA. In this draft policy documents are included technical and scientific elements that constitute basic information and basic sciences related to the concept and application of the new method of study and monitoring of the behavior of eukaryotic cells. The scope and depth of this new method of cancer research (RIBOGRAMA) is explained in brief monograph on the concept and its applications, which is part of the work of introduction and dissemination of technical and scientific potential of this innovative method as the monitoring of modification of the phenotype of eukaryotic cells to stimuli that face the medium in which they are provoked.

1- APPROACH TO THE TOPIC In this initial approach is intended to make a brief introduction about the method RIBOGRAMA and their practical utility in screening, diagnosis and treatment of cancer in any organ. We'll start by talking in the case of cancer of the rectum and colon, as one of the cancers with highest incidence in the population of the Western type diet. The method is based on a new biomolecular technology (noninvasive), which allows the very early diagnosis of cancerous lesions of the colon and rectum. We begin by making the presentation of the method RIBOGRAMA on colon cancer and rectum, presenting its concept and clinical applications.

This new method allows a diagnosis stage "pre-carcinoma in situ", since it is based on the phenotype of the cells of mucosa of the colon and rectum, comprising very high amounts of free ribosomes in the cytoplasm, which can be quantified by using flow cytometry after their isolation, and marked with fluorochromes. The amounts of these registers repeated ribosomes establish a graphical curve (RIBOGRAMA) which represents the degree of malignancy of a tendency that above a certain level / concentration value can mean that the cells of a tissue (eg. of colon and rectum) can be considered to be in the process of developing a carcinoma, before any macroscopic observation (endoscopy). This means that we have a new method, on a clear biomolecular level, as demonstrated in the above Doctoral Thesis, with advantages over macroscopic observation (endoscopy). This new method is much greater scope than has been achieved in other noninvasive methods of studying cancer of the colon and rectum (test for fecal occult blood testing and the DNA mutations in the stool).

Thanks to this method, it is possible to create a study/screening, with a non-invasive use, with great acceptance by the population, in which every citizen to submit a curve of high RIBOGRAMA, must necessarily be observed through a diagnostic endoscopy and treatment (at the same time and remain under observation therapy and prevention of the appearance of any new lesions by a special diet and treatment with acetylsalicylic acid and antiinflammatory – nonsteroidal, with proven efficacy in preventing and reducing polyp formation or any injury-prone pre-malignancy. This represents huge savings for the investment therapeutic cancer.

2 – OVERVIEW It is intended to start a project related to the creation of a new noninvasive test for very early diagnosis of cancer of the colon and rectum initially (and then other organs and systems, later). The RIBOGRAMA is a new technique that results from a research on cancer of the colon and rectum (CRC), in the Complutense 8

RIBOGRAMA PROJECT University of Madrid, for the design and creation of a stool collection kit and the study and calculating the quantities of free ribosomes in a number of colonocytes, which allow to make the diagnosis of CRC very early, well before the execution of a colonoscopy in order to detect pre-malignant lesions. The aim of the research considers primarily the following steps: a) Creating a device for collecting feces that contain millions of cells released from the mucosa of the colon and rectum - called colonocytes, which are to cover the surface of the stool; and dipped in a preserving serum of colonocytes; b) Separation of cells (colonocytes) stool; c) Calibration and standardization of colonocytes by rupture, by using certain routine techniques; d) Isolation of ribosomes by differential centrifugation; e) Marking of ribosomes through the use of fluorochromes (nanotechnology); f) Count of ribosomes, using flow cytometry the reverse-transcriptase qPCR or RT-qPCR. The ideas and concepts developed in this research are already legally protected intellectually and patented by a patenting company in Madrid. It is intended to enter into a contract of partnership with one or more investors, by creating a company that can develop a diagnostic kit handy and easily accessible to the general public. Kit was created that will constitute a collecting feces, since the diagnostic test is performed by counting/ quantitation of contained free ribosomes in the cytoplasm of cells from mucosa of the colon and rectum, the intestinal wall desquamated, which are covering the surface of the stool eliminated.

3 - ESSENCE OF THE METHOD The readings results related to the quantities of free ribosomes observed in the samples of the cells under study are recorded on a chart corresponding to two variables: - the time variable entered in the horizontal axis of the graph, and variable amounts of ribosomes entered in the vertical axis graph. With these two variables it is possible to construct a graphically curve. The variable quantity represents the mean density of the ribosomes in the cytoplasm of every cell and variable time corresponds to the time interval at which to observe variations of the amount/average number of ribosomes per cell. This project will create a profile graph of numerical values corresponding to an average normal that would correspond to the average amount of ribosomes in cells considered normal. From then on, it is possible to do comparative studies with other graphic curves deviated from the normal curve, which reflect the tendencies of potential malignancy (by significant elevation RIBOGRAMA curves corresponding to a community of cells under investigation and in a process of malignization.

These descriptions of the variation curves correspond to RIBOGRAMA trend patterns of behavior of malignant cells/tissues, well ahead of the microscopic study of biopsy specimens of these tissues reveal malignancy. The application of this method, based on molecular biology techniques, allows early information about the behavior of cell phenotype under study. This shows a marked increase in sensitivity to detect very early malignant tissue, allowing their use in diagnosis and screening for pre-cancerous disease. The curve of RIBOGRAMA, as shown, has a mathematical base (allowing be reproduced in comparable circumstances), that is, there is a correspondence to a language non-subjective. In other words, the analysis of the trend curves of RIBOGRAMA corresponds to a marked indicator of the degree of development and propensity towards the malignancy, through a process of monitoring a chronological time. We are thus faced with a rigorous new method, allowing comparison studies on the effects of stimuli on cells.

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RIBOGRAMA PROJECT 4 - SCOPE AND POTENTIAL OF THE METHOD Cancer of the colon and rectum is one of the highest incidences of human cancer in countries with lifestyle and food cooked in the Western type. This research project has strong potential for public health, hospital and medical practice in clinical and laboratory research in the food industry, veterinary medicine, in cancer research, in the pharmaceutical industry and clinical trials, monitoring of ecological systems, the studding the pollution in nature, etc., with great medical and financial impact, first, by far, the significant lowering morbidity and mortality rates of colorectal cancer, worldwide, and then to other organs and systems.

RIBOGRAMA is an objective and quantitative method that can provides a unique service to health care providers worldwide. The method is designed to run transversely from the clinical practice of office to the largest chain of hospitals. The RIBOGRAMA method is prepared be presented and recommended in many challenges to providers of health services in today's business world in ever-changing environments, for biomedical activity and life sciences in general and, in biosciences, also offers some basic common utility in a global perspective of the worldwide "bio-industries'. The timing for this method to enter the domestic and global markets is understandable and current. This method presents itself as a great solution for people and countries across the world to help control the cancer disease.

5 - MOTIVATION AND ARGUMENTS FOR USING THE METHOD 1. The CRC is the leading cause of cancer death in the world with Western lifestyle, but is within the number of cancer with better healing potential, when identified at an early stage; 2. This cancer has a long premalignant phase and a slow progression from the stage of the disease confined to the wall of the organ to the stages of local invasion and distant metastatic disease.

Therefore, there is ample opportunity to identify patients in a pair of stage curable disease by conducting extensive surveys; 3. In a study of CRC screening were explained the advantages and disadvantages of various methods of tests conducted on 100 people, and then was asked what methods people prefer as a method of screening. The results were that one third of patients declined any form of invasive test, but that would undergo a non-invasive test; 4. The screening of the CRC proved to be effective, both in reducing mortality and decline in incidence; 5. There are certain circumstances and conditions that decrease the potential of the results of the surveys, which are associated with various factors, such as: a) The number and type of screening strategies; b) The various aspects of recommendation from improper handling and inadequate testing of fecal occult blood; c) Excessive time and preparation uncomfortable because the patient has to do prior bowel cleansing; d) Nature of many invasive and rigorous screening procedures.

6. All these factors contribute to lower benefits and lower the value of the survey effort; 7. The screening of CRC did not penetrate adequately in population samples capable of being studied, the indications for this, as, p. ex. above the 40/50 years of age; 8. It is necessary to create other systems for clinical screening to ensure that there are patients who rely on tight screening programs, which can produce the conditions for implementation within the complexity of motivating people, in adherence to screening.

The RIBOGRAMA method allows patients in a quantifiable manner, non-invasive, easy adhesion of the stocks, through the study of amount/density of ribosomes in colorectal mucosal cells, which have been mixed 10

RIBOGRAMA PROJECT with faeces. The collection becomes possible with the use of a faecal collector, which can fit a toilet of any person who opts for this test, do not need to manipulate their own feces, such as in occult blood test.

It is seen, therefore, that the method gives a concrete numerical result, is convincing to the patient and his family, as well as the people with whom he contacts the day-to-day induction causing them to also join the test making CRC treatable and controllable. With this method the patient can buy a Kit/collector stools, accompanied by a small book of illustrated instructions (see your design and design at the end of this brief summary of the method of promotion RIBOGRAMA, at a pharmacy, or a shopping center, which leads to home in the privacy of his home, to collect the biological sample of colonocytes, after making the delivery of the product, properly preserved and packaged in a laboratory certified for testing. The practical consequences of using this method is that it will push down, in a very significant way, the costs in the area of health expenditures, resulting from the treatment of colorectal cancer in a very early stage, with excellent conditions to meet precancerous lesions (polyps) which, once removed, they no longer cause danger of malignancy. With the RIBOGRAMA test people know when they should go to a doctor because they have the control/monitoring of the RIBOGRAMA curve.

The RIBOGRAMA curve gives a quantified information on the risk of occurrence (or not) of lesions like polyp, reflecting the possibility of a malignant process if values are reached, when the concentration of free ribosomes are above the numerical value established as normal. Thus, it becomes unnecessary to make a "blind" colonoscopy, without any utility and convenience to the patient, since only the lesions and/or tissues with high levels (above a certain threshold) of free ribosomes are amenable to may eventually develop a malignant process. This new method provides, for the first time, throughout the scientific world community, the ability to identify, safely, when a tissue is undergoing malignant phenomena, which may be interrupted by a correction with a specific diet and habits, as well as the concurrent use of aspirin and nonsteroidal antiinflammatory – steroids, and other drugs, which together cause the inversion phenomenon of the malignant polyps. In other words, we are faced with a secure monitoring and control of the phenomenon of malignancy at extremely low prices. This means that in a scenario of large masses of the population, with reasonable patient adherence, undergoing this non-invasive test, it can promote significant amount of financial savings, in almost astronomical figures. This method, as mentioned above, may be extended to other organs.

For example, it should be noted that the RIBOGRAMA method can be repeated, at low cost, during short time intervals, without there being the risk of being surprised at the onset of a malignant lesion, accessible to any patient, without needing to undergo the uncertainty of the result of colonoscopy with their discomfort, their prior preparation, to bowel cleansing enemas and use of cathartics, with the usual abdominal cramps, besides the high costs of implementing a colonoscopy. Speaking of costs, with the CRC disease, once carried the diagnosis of malignancy, that can find a cancerous lesion exceeding the limit of the submucosa, can speak in multiple financial burdens as a result of colorectal cancer, which can enumerate up the following: 1.

2.

3.

4.

5.

6.

Charges related to inpatient hospitalization; Charges related to the use of the operating room; Charges related to intensive care and / or recovery room for surgical patients; Costs related to chemotherapy and other treatments such as radiotherapy; Charges related to multiple and repeated diagnostic procedures in the follow-up of the patient; Charges related to multiple and various medications and therapies and the repeated diagnostic procedures, at the follow-up of the patient; 7. Costs related to the Social Security System, in which the patient with colorectal cancer is no longer active contributor to the system, would become recipient/beneficiary of subsidies in the duration of the disease;

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RIBOGRAMA PROJECT 8. Usually the patient will not go alone to any of these interventions in the area of oncology in their health, being accompanied by a family member or close friend, what this represents an annual loss of millions of hours of work for the community where they live.

This project has the potential to generate strong and sustainable economic and financial return within 5 years.

A. Ferreira-Alemao, MD PhD UNIVERSIDAD COMPLUTENSE DE MADRID

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III - TRADITIONAL (ACTUAL) COLORECTAL CANCER SCREENING Traditional Methods of Colorectal Screening: Advantages, Problems, and Comparisons Screening for colon cancer decreases both the incidence and the death rate of the disease by detecting and leading to the removal of precancerous adenomatous polyps (growths that develop on the membrane of the large intestine) before they become invasive cancers (1-8). Health care providers and policy makers agree that screening for colorectal cancer is worthwhile (1). The current options available for colorectal cancer screening include digital rectal examination, fecal occult blood testing, barium enema, sigmoidoscopy, fiberoptic colonoscopy, or any combination of these procedures (8). Following is a brief description of current colorectal screening method: Digital rectal examination: The physician inserts a finger into the rectum to feel for polyps or other abnormalities.

Fecal occult blood testing (FOBT): A sample of feces is tested for microscopic amounts of blood. This could be an indication of a bleeding tumor, although there are many other causes of blood in the stool as well.

Barium enema: A suspension of barium sulfate is injected through the anus, which coats the rectum and colon and makes these areas appear opaque on an x-ray. When an x-ray is subsequently taken, abnormalities can be seen. In a double contrast barium enema, air is pumped into the rectum after the initial x-ray and the x-ray is repeated.

Sigmoidoscopy: In this procedure a sigmoidoscope, which is a thin, lighted tube with a camera at the end, is inserted into the rectum and guided into the sigmoid colon (the lower portion of the colon). This test reveals polyps, tumors, and other abnormalities in the rectum and sigmoid colon.

Fiberoptic colonoscopy: In this test, which requires sedation, the lining of the entire colon and rectum is examined. Like a sigmoidoscopy, the test is performed with a thin, lighted tube with a camera attached, but in a colonoscopy, the tube is guided further into the colon to visualize the entire colon.

The colonoscope pumps air into the colon, while the video camera records the images on a screen for the doctor to see during the procedure. If polyps or other abnormal tissue are discovered, they can be removed and biopsied.

The following strategies have been recommended by The United States Agency for Health Care Policy and Research for colon screening in the average risk patient (3). Fecal occult blood testing (FOBT) is recommended every year beginning at age 50. If this test is positive at any year, a diagnostic exam should be performed including either colonoscopy or double contrast barium enema (possibly in combination with sigmoidoscopy) (9). For individuals with no symptoms, either flexible sigmoidoscopy or double contrast barium enema should be performed every five years or colonoscopy every 10 years for the detection of non-bleeding polyps and tumors (3, 4).

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RIBOGRAMA PROJECT Despite consensus on the need for and efficacy of screening, there consistently are approximately 60,000 deaths from colon cancer every year in the United States (2). Since most colorectal cancers grow slowly from precancerous adenomas, and screening can detect the precancerous adenomas, the continued high prevalence of colon cancer is alarming. There are many potential reasons for the high rate of colon cancer, including limitations of current screening options, confusion about when and how to perform current screening options, and patient reluctance to undergo screening.

Regarding patient reluctance to comply with current screening options, a survey found that only 17.3 percent of patients over age 50 had undergone fecal occult blood testing within the last year and only 9.4 percent had undergone sigmoidoscopy within the last three years (9). Studies have also demonstrated that even health care professionals are reluctant to undergo colon screening (10). The Centers for Disease Control has set goals and committed funding to decrease the number of deaths from colorectal cancer. Among the strategic goals is to increase the number of patients accessing colorectal cancer screening from the current levels of approximately 37 percent to 50 percent by the year 2010 (http://www.cdc.gov/cancer/screenforlife/index.htm).

Each current colon screening option has important limitations. While the performance of yearly FOBT has demonstrated a mortality reduction from colorectal cancer, FOBT does not directly evaluate mucous membrane of the colon. (11). Many large adenomatous polyps do not bleed and occasionally cancers will not bleed. In addition, there are many false positive fecal occult blood tests for colon cancer, which can lead to further testing and expense. A study demonstrated that in more than 50 percent of stool examinations in which blood is found, the source was from the upper gastrointestinal tract (12).

Screening sigmoidoscopy has been shown to decrease the death rate of colorectal cancer (13). However, sigmoidoscopy fails to evaluate the entire colon and therefore complete colon screening is not obtained (14, 15). Recent studies evaluating sigmoidoscopy and colonoscopy found that if only sigmoidoscopy were performed for colon screening in people with no symptoms, many advanced cancers in the upper colon would be missed (14, 15). This is true even taking into account the fact that if a significant lesion were detected in the part of the colon examined during sigmoidoscopy, it would prompt a complete colon examination with colonoscopy. In fact, half of the cases of upper-colon cancers did not have a lower-colon polyp which would have prompted colonoscopy. Moreover, it appears that the combination of FOBT and sigmoidoscopy do not result in a significant improvement in the efficacy of screening (16).

There are currently two options for a full colonic evaluation: colonoscopy and double contrast barium enema (DCBE). The sensitivity of the double contrast barium enema for polyp detection is incompletely documented.

Norfleet et al determined that the sensitivity of DCBE was 26 percent in detecting polyps larger than 5 mm as compared to 13 percent for single contrast barium enema (17). Importantly, a recent study comparing double contrast barium enema with colonoscopy in detecting polyps in patients who previously had a polyp removed demonstrated poor sensitivity of the double contrast barium enema. In this study the barium enema missed over 50 percent of polyps greater than 1 cm in diameter (18).

Of the numerous options for colon cancer screening, complete fiberoptic colonoscopy allows the most thorough evaluation of the colon with the added benefit that suspicious lesions can be excised during the exam and then biopsied. Colonoscopy is considered the "gold standard" for colonic evaluation (1,19). Yet there are many limitations to the widespread use of colonoscopy for screening, including examination time, need for sedation, potential risk of perforation and bleeding, costs of the procedure including the need for sedation, failure to complete the examination in 5-10 percent of patients, and an insufficient workforce of trained endoscopists (physicians trained to perform internal visualization procedures such as colonoscopies) to meet the increased demand (20, 21).

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RIBOGRAMA PROJECT A final limitation of conventional fiberoptic colonoscopy is that in order to perform the examination, the colon must be thoroughly cleansed of residual fecal material. This is typically performed with polyethylene-glycol solutions or phospho-soda preparations. Many patients find bowel cleansing the most difficult aspect of screening, whether sigmoidoscopy, DCBE, or traditional colonoscopy, or virtual colonoscopy is used (22, 23, 24).

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References: 1. 1. Ransohoff DF and Sandler RS. Clinical Practice: Screening for colorectal cancer. NEJM 2002; 346:40-44.

2. 2. Parker SH, Torry T, Bolden S and Windigo PA. Cancer statistics 1996. CA-Cancer J Clin 1996;65:5-27.

3. 3. Glick S, Wagner JL and Johnson CD. Cost-effectiveness of double contrast barium enema in screening for colorectal cancer. AJR 1998; 170:629-636.

4. 4. Winawer S, Fletcher R, Rex D, et al. Colorectal cancer screeningand surveillance: clinical guidelines and rationale-update based on new evidence. Gastroenterology 2003; 124:544-560.

5. 5. Mandel JS, Bond JH, Church TR, et al. Reducing the mortality from colorectal cancer by screening for fecal occult blood. N Engl J Med 1993; 328:1365-1371.

6. 6. Winawer SJ, Zauber AG and Ho MN. Prevention of colorectal cancer by colonoscopic polypectomy. The National Polyp Study Workgroup. NEJM 1993; 329:1977-1981.

7. 7. Muller AD and Sonnenberg A. Protection by endoscopy against death from colorectal cancer. A case control study among veterans. Arch Int Med 1995; 155:1741-1748.

8. 8. Eddy DM. Screening for colorectal cancer. Ann Intern Med 1990; 113:373-384.

9. 9. Winawer SJ, Fletcher RH, Miller l, et al. Colorectal cancer screening: clinical guidelines and rationale. Gastroenterology 1997; 112:594-601.

10. 10. Vining DJ. Virtual endoscopy: is it reality. Radiology 1996; 200:30-31.

11. 11. Kronborg O, Fenger C, Olsen J, et al. Randomized study of screening for colorectal cancer with fecal occult blood test. Lancet 1996; 348:1467-1471.

12. 12. Rockey DC, Koch J, Cello JP, Sanders LL and McQuaid K. Relative frequency of upper gastrointestinal and colonic lesions in patients with positive fecal occult-blood tests. NEJM 1998; 339:153-159.

13. 13. Selby JV, Friedman GD, Quesenberry PC Jr. and Weiss NS. A case control study of screening sigmoidoscopy and mortality from colorectal cancer. N Engl J Med 1992; 326:653-657.

14. 14. Lieberman DA, Weiss DG, Bond JH, et al. Use of colonoscopy to screen asymptomatic adults for colorectal cancer. NEJM 2000; 343:162-168.

15. 15. Imperiale TF, Wagner DR, Lin CY. Risk of advanced neoplasms in asymptomatic adults according to the distal colorectal findings. NEJM 2000; 343:169-174.

16. 16. Podolsky DK. Going the distance-the case for true colorectal-cancer screening. NEJM 2000; 343:207-208.

17. 17. Norfleet RG, Ryan ME, Wyman JB, et al. Barium enema versus colonoscopy for patients with polyps found during flexible sigmoidoscopy. Gastrointest Endosc 1991; 37:531-534.

18. 18. Winawer SJ. Stewart ET. Zauber AG. A comparison of colonoscopy and double-contrast barium enema for surveillance after polypectomy. NEJM 2000; 342:1766-1777.

19. 19. Liberman DA and Weiss DG. One time screening for colorectal cancer with combined fecal occult-blood testing and examination of the distal colon. NEJM 2001; 345:555-560.

20. 20. Anderson ML, Heigh RI, McCoy GA, et al. Accuracy of assessment of the extent of examination by experienced colonoscopists. Gastrointest Endosc 1992; 38:560-563.

21. 21. Detsky AS. Screening for colon cancer-can we afford colonoscopy? NEJM 2001; 345:607-608.

22. 22. Ristvedt SL, McFarland EG, Weinstock LB, Thyssen EP. Patient preferences for CT colonography, conventional colonoscopy, and bowel preparation. Am J Gastroenterol 2003; 98:578585.

23. 23. Gluecker TM, Johnson CD, Harmsen WS, et al. Colorectal cancer screening with CT colonography, colonoscopy, and double contrast barium enema examination: prospective assessment of patient perceptions and preferences. Radiology 2003; 227;378-384.

24. 24. Weitzman ER, Zapka J, Estabrook B, Goins KV. Risk and reluctance: understanding impediments to colorectal cancer screening. Prev Med 2001; 32:502-513.

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RIBOGRAMA PROJECT

IV - STATISTICAL DATA ON COLORECTAL CANCER (CRC) 1 - FECAL OCCULT BLOOD TEST (FOBT) In FOBT testing, a person collects stool samples that are analyzed for the presence of small amounts of blood.

Collection details vary somewhat for different tests, but typically involve collection of as many as three different specimens on 3 different days, with small amounts from one specimen smeared by a wooden stick on a card with two windows or otherwise placed in a specimen container. The guaiac test identifies peroxidase-like activity that is characteristic of human and nonhuman hemoglobin. Thus, it will record blood from ingested meat, upper airway bleeding such as epistaxis, upper gastrointestinal (GI) bleeding, as well as colonic lesions.

Five controlled clinical trials have been completed or are in progress to evaluate the efficacy of screening utilizing the FOBT. The Swedish trial is a targeted study for individuals aged 60 to 64 years.(1) The English trial selects candidates from lists of family practitioners.(2) The Danish trial offers screening to a population aged 45 to 75 years randomly assigned to a control or study group.(3,4) The Minnesota trial demonstrated that annual guaiac-based FOBT (gFOBT) testing using primarily rehydrated samples decreased mortality from colorectal cancer (CRC) by 33% (5) and that biennial testing developed a 21% relative mortality reduction.(6) Some part of the reduction may have been attributed to chance detection of cancer by colonoscopies; rehydration of guaiac test slides greatly increased positivity and consequently increased the number of colonoscopies performed.(7) Subsequent analyses by the Minnesota investigators using mathematical modeling suggested that for 75% to 84% of the patients mortality reduction was achieved because of sensitive detection of CRCs by the test; chance detection played a modest role (16%–25% of the reduction).(8) Nearly 85% of patients with a positive test underwent diagnostic procedures that included colonoscopy or double-contrast barium enema plus flexible sigmoidoscopy (FS). After 18 years of follow-up, the incidence of CRC was reduced by 20% in the annually screened arm and 17% in the biennially screened arm.(9) The English trial allocated approximately 76,000 individuals to each arm. Those in the screened arm were offered nonrehydrated gFOBT testing every 2 years for three to six rounds from 1985 to 1995. At a median follow-up time of 7.8 years, 60% completed at least one test, and 38% completed all tests. Cumulative incidence of CRC was similar in both arms, and the trial reported a relative risk (RR) reduction of 15% in CRC mortality (odds ratio [OR] = 0.85; 95% confidence interval [CI], 0.74–0.98).(10) The serious complication rate of colonoscopy was 0.5%. There were five deaths within 30 days of surgery for screen-detected CRC or adenoma in a total of 75,253 individuals screened.(11) After a median follow-up of 11.8 years, no difference in CRC incidence between the intervention and control groups was observed. The disease-specific mortality rate ratio associated with screening was 0.87 (0.78–0.97; P = .01). The rate ratio for death from all causes was 1.00 (0.98–1.02; P = .79).(12) When the median follow-up was extended to 19.5 years, there was a 9% reduction in CRC mortality (RR = 0.91; 95% CI, 0.84–0.98) but no reduction in CRC incidence (RR = 0.97; 95% CI, 0.91–1.03), or death from all causes (RR = 1.00; 95% CI, 0.99–1.02).(13) The Danish trial in Funen, Denmark, entered approximately 31,000 individuals into two arms, in which individuals in the screened arm were offered nonrehydrated gFOBT testing every 2 years for nine rounds over a 17-year period. Sixty-seven percent completed the first screen, and more than 90% of individuals invited to each subsequent screen underwent FOBT testing. This trial demonstrated an 18% reduction in CRC mortality at 10 years of follow-up,(12) 15% at 13 years of follow-up (RR = 0.85; 95% CI, 0.73–1.00),(15) and 11% at 17 17

RIBOGRAMA PROJECT years of follow-up (RR = 0.89; 95% CI, 0.78–1.01).(16) CRC incidence and overall mortality were virtually identical in both arms.

The Swedish trial in Goteborg enrolled all 68,308 citizens in the city born between 1918 and 1931 that were aged 60 to 64 years, and randomly assigned them to screening and control groups of nearly equal size.

Participants in the control group were not contacted and were unaware they were part of the trial. Screening was offered at different frequencies to three different cohorts according to year of birth. Screening was done using the gFOBT Hemoccult-II test after dietary restriction. Nearly 92% of tests were rehydrated. Individuals with a positive test result were invited to an examination consisting of a case history, FS, and double-contrast barium enema. The range of follow-up times was from 6 years 7 months to 19 years 5 months, depending on the date of enrollment. The primary endpoint was CRC-specific mortality. The overall screening compliance rate was 70%, and 47.2% of participants completed all screenings. Of the 2,180 participants with a positive test, 1,890 (86.7%) underwent a complete diagnostic evaluation with 104 cancers and 305 adenomas of at least 10 mm detected. In total, there were 721 CRCs (152 Dukes D, 184 Dukes C) in the screening group and 754 CRCs (161 Dukes D, 221 Dukes C) in the control group, with an incidence ratio of 0.96 (95% CI, 0.86– 1.06). Deaths from CRC were 252 in the screening group and 300 in the control group, with a mortality ratio of 0.84 (95% CI, 0.71–0.99). This CRC mortality difference emerged after 9 years of follow-up. Deaths from all causes were very similar in the two groups, with a mortality ratio of 1.02 (95% CI, 0.99–1.06).(1) All trials have shown a more favorable stage distribution in the screened population compared with controls.

Data from the Danish trial indicate that while the cumulative incidence of CRC was similar in the screened and control groups, a higher percentage of CRCs and adenomas were Dukes A and B lesions in the screened group.(14) A meta-analysis of all previously reported randomized trials using biennial FOBT showed no overall mortality reduction by gFOBT screening (RR = 1.002; 95% CI, 0.989–1.085). The RR of CRC death in the gFOBT arm was 0.87 (95% CI, 0.8–0.95), and the RR of non-CRC death in the gFOBT group was 1.02 (95% CI, 1.00–1.04; P = .015).(17) Mathematical models have been constructed to extrapolate the results of screening trials to screening programs for the general population in community health care delivery settings. These models project a reduction in CRC mortality or an increase in life expectancy using currently available screening methodology.(18-21) The anticipated success of such methodology is critically dependent on the appropriate use of the FOBT and an effective clinical management plan.(22,23) A systematic review done through the Cochrane Collaboration examined all CRC screening randomized trials that involved gFOBT testing on more than one occasion. The combined results showed that trial participants allocated to screening had a 16% lower CRC mortality (RR = 0.84; 95% CI, 0.78–0.90). There was, however, no difference in all-cause mortality between the screened and control groups (RR = 1.00; 95% CI, 0.99–1.02).

Furthermore, the trials reported a low positive predictive value (PPV) for the FOBT test, suggesting that most positive tests were false positives. From the trials with nonrehydrated slides (Funen and Nottingham), the PPV was 5.0% to 18.7%, while the PPV in the trials using rehydrated slides (Goteborg and Minnesota) was 0.9% to 6.1%. The report contains no discussion on contamination in the control arms of the trials and no information on treatment by stage.(24,25) On initial (prevalence) examinations, from 1% to 5% of unselected persons tested with gFOBT have positive test results. Of those persons with positive test results, approximately 2% to 10% have cancer and approximately 20% to 30% have adenomas,(26,27) depending on how the test is done.

NEWER FOBTS: NONRANDOMIZED CONTROLLED TRIAL EVIDENCE The immunochemical FOBT (iFOBT) was developed to detect intact human hemoglobin. The advantage of iFOBT over guaiac FOBT (gFOBT) is that it does not detect hemoglobin from nonhuman dietary sources. It also does not detect partly digested human hemoglobin that comes from the upper respiratory or GI tract.

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RIBOGRAMA PROJECT Preliminary studies of several commercially developed iFOBT tests define their sensitivity and specificity compared with concurrently performed colonoscopy. These studies also examine these outcomes for different cut points, and the benefit of multiple versus single stool samples.(29) Generally, iFOBT testing is more sensitive for cancers than for benign neoplasias. As expected, higher cut points decrease sensitivity and increase specificity.

In one study, 2,188 patients scheduled for colonoscopy because of an elevated risk due to personal or family history of colorectal neoplasm, positive gFOBT result, change in bowel habits, anemia, abdominal pain with weight loss, or anal symptoms were invited to participate in a comparative assessment of iFOBT against colonoscopy findings. After exclusions for health and cognitive reasons, 1,859 patients were offered iFOBT, 1,116 patients adhered to the protocol, and 1,000 patients completed the procedure. Sensitivity and specificity were calculated at various cut-points. At a cut-point of 100 ng/mL, sensitivity and specificity were, respectively, 88.2% and 89.7% for cancer and 61.5% and 91.4% for any clinically significant neoplasia (cancer and advanced polyps). At 150 ng/mL the respective sensitivities and specificities were 82.4% and 91.9% for cancer and 53.8% and 95% for any clinically significant neoplasia. Calculations were based on the most severe pathologic finding from colonoscopy and the highest fecal-hemoglobin concentration measured by iFOBT applied to three stool samples collected prior to the colonoscopy. Stool samples were collected by patients following iFOBT kit instructions and analyzed by the OC-MICRO analyzer (from the Eiken Chemical Company in Tokyo, Japan).(30) In another study, 21,805 asymptomatic patients received iFOBT based on one stool sample collected by patients following the kit instructions on the day of or the day before the colonoscopy. Stool samples were analyzed using the Magstream 1,000/Hem SP automated system (from Fujirebio Incorporated, Tokyo, Japan), which is based on the HemeSelect system (from Beckman Coulter, Palo Alto, California). Sensitivity and specificity based on subsequent colonoscopy were, respectively, 65.8% and 94.6% for cancer and 27.1% and 95.1% for advanced neoplasm.(31) Fecal immunochemical tests may vary with regard to numbers of stools tested and cut-off values for a positive result.(29,32,33) A systematic review to evaluate the comparative diagnostic performance of gFOBT and iFOBT in the context of a decision to introduce screening for CRC in the United Kingdom, included 33 studies evaluating gFOBT and 35 studies evaluating iFOBT, including nine that evaluated both gFOBT and iFOBT. There was no clear evidence for superiority of either gFOBT or iFOBT. Sensitivities for the detection of all neoplasms ranged from 6.2% (specificity 98%) to 83.3% (specificity 98.4%) for gFOBTs and 5.4% (specificity 98.5%) to 62.6% (specificity 94.3%) for iFOBT. Increasing sensitivity entailed adjusting cut-points to decrease specificity. Sensitivities were higher for the detection of CRC and lower for adenomas.(34) Some studies have utilized the quantitative ability of iFOBT to consider detection and specificity at various test cut-points for defining a positive test. One study (35) found that reducing the cut-point from the standard 100 ng/mL to 50 ng/mL increased the detection of advanced adenomas but had little impact on the detection of cancer. The number of colonoscopies required to detect a single advanced adenoma or cancer increased from 1.9 to 2.3; a 20% increase. Specificity declined from 97.8% to 96%.

Potential false-positive test results due to an increased risk of upper GI bleeding are of concern with FOBT testing and pretest protocols, therefore; low-dose aspirin regimens should be discontinued for a week or more prior to FOBT. The performance of iFOBT was tested in an ongoing diagnostic study (2005–2009) at 20 internal medicine GI practices in southern Germany. Nineteen hundred seventy-nine patients (233 regular lowdose aspirin users and 1,746 never users) were identified in the records for inclusion in the analysis. All patients provided one stool sample taken within a week before colonoscopy preparation, which was collected according to instructions in a container that was kept refrigerated or frozen until rendered to the clinic on the day of colonoscopy, and the patients agreed to complete a standard questionnaire regarding the use of analgesics and low-dose aspirin (for prevention of cardiovascular disease). Stool samples were thawed within 19

RIBOGRAMA PROJECT a median of 4 days after arrival at the central laboratory (shipped frozen from the recipient clinics). Fecal occult blood levels were measured by two automated iFOBT tests according to the manufacturer’s instructions (RIDASCREEN Haemoglobin and RIDASCREEN Haemo-/Haptoglobin Complex, r-biopharm, Bensheim, Germany) following clinical procedures and blinded to colonoscopy results. Advanced neoplasms were found in 24 aspirin users (10.3%) and in 181 nonusers (10.4%). At the cut-point recommended by the manufacturer, sensitivities for the two tests were 70.8% (95% CI, 48.9%–87.4%) for users compared with 35.9% (95% CI, 28.9%–43.4%) for nonusers and 58.3% (95% CI, 36.6%–77.9%) for users compared with 32% (95% CI, 25.3%–39.4%) for nonusers (P = .001 and P = .01, respectively). Specificities were 85.7% (95% CI, 80.2– 90.1%) for users compared with 89.2% (95% CI, 87.6%–90.7%) for nonusers and 85.7% (95% CI, 80.2%– 90.1%) for users compared with 91.1% (95% CI, 89.5%–92.4%) for nonusers (P = .13 and P = .01, respectively). For these iFOBTs, sensitivity for advanced neoplasms was notably higher with the use of lowdose aspirin while specificity was only slightly reduced, suggesting that there might be an advantage to aspirin use to increase sensitivity without much decrease in specificity.(36)

2 – SIGMOIDOSCOPY The flexible fiberoptic sigmoidoscope was introduced in 1969. The 60 cm flexible sigmoidoscope became available in 1976.(37) The flexible sigmoidoscope permits a more complete examination of the distal colon with more acceptable patient tolerance than the older rigid sigmoidoscope. The rigid instrument can discover 25% of polyps, and the 60 cm scope can find as many as 65%. The finding of an adenoma by FS may warrant colonoscopy to evaluate the more proximal portion of the colon.(38,39) The prevalence of advanced proximal neoplasia is increased in patients with a villous or tubulovillous adenoma distally and is also increased in those aged 65 years or older with a positive family history of CRC and with multiple distal adenomas.(40) Most of these adenomas are polypoid, flat and depressed lesions, which may be somewhat more prevalent than previously recognized.(41) Virtually all screening studies using these types of sigmoidoscopes have demonstrated an increase in the proportion of early cases and a corresponding increase in survival compared with cases diagnosed in a nonscreening environment. Most of these studies, however, lack appropriate comparison groups, and their interpretation is unclear because of screening biases.

The first incidence and mortality results from a randomized trial of sigmoidoscopy were reported in the Norwegian Colorectal Cancer Prevention (NORCCAP) trial. This trial randomly assigned 41,913 men and women aged 55 to 64 years to a usual-care control group and 13,823 individuals to a screening group, 6,915 of whom received one-time FS and 6,908 of whom received both FS and iFOBT. Screening was conducted from January 1999 to December 2000. Follow-up was through national registries in Norway and ended December 31, 2006 for incidence and December 31, 2005 for mortality. The primary endpoint was incidence of CRC after 5, 10, and 15 years of follow-up based on an intention-to-screen analysis. The two randomly assigned groups had the same median age of 59 years and equal gender distribution. The attendance rate for screening was 65%. Mean insertion depth of the endoscope was 48.9 cm for men and 44.0 cm for women, with no severe complications. A neoplastic lesion was found in 19% of people screened, and 5% had a highrisk adenoma or invasive cancer. There were 33 prevalent CRCs, 17 in the subgroup invited for sigmoidoscopy only and 16 in the subgroup invited to both tests. Compliance for colonoscopy work-up was 97%. There was no difference in the cumulative hazard of CRC between the screened and control groups; 134.5 versus 131.9 cases per 100,000 person years, after a median follow-up of 7 years. The hazard ratio (HR) for CRC mortality was 0.73 (95% CI, 0.47–1.13) and for rectosigmoidal cancer mortality was 0.63 (95% CI, 0.34–1.18) after a median follow-up of 6 years. The HR for all-cause mortality was 1.02 (95% CI, 0.98– 1.07). Additional follow-up is planned.(42) 20

RIBOGRAMA PROJECT In the Telemark Study in Norway, 400 people aged 50 to 59 years were offered FS in 1983 and, if polyps were found, a colonoscopy was available to them at that time, and again 2 years later, and also 6 years later (the intervention was more aggressive than the one-time sigmoidoscopy currently being tested in the United Kingdom). A nonrandomized control group was selected that did not have a baseline examination. In 1996, all persons were invited to have a colonoscopy. The result was that ten persons in the control group and two in the screening group developed CRC (RR = 0.2; 95% CI, 0.03–0.95). The screening group, however, had a higher overall mortality compared with the control group.(43) A RCT of sigmoidoscopy screening in the United Kingdom suggests that the impact of endoscopic screening, at least on the left side of the colon, is substantial and prolonged. In this RCT, 170,000 persons were randomly assigned to one-time sigmoidoscopy versus usual care. At sigmoidoscopy, polyps were removed and patients with cancer were referred for treatment. Based on sigmoidoscopy findings, persons were considered to be low risk if they had normal exams or only one or two small (<1 cm) tubular adenomas; such persons were not referred for either colonoscopy workup or colonoscopic surveillance. In a follow-up of 10 years, the left-sided CRC incidence in the low-risk group (about 95% of attendees were low risk) was 0.02% to 0.04% per year—a very low risk of CRC compared with average risk. The cause of reduced risk—whether due to detection and removal of large or small polyps, or selection of individuals at lower risk—is yet unclear,(44) but may be assessed in further analysis. The natural history of large polyps is not well known, but some evidence suggests that such lesions become clinical CRC at a rate of approximately 1% per year.(45) Evidence from multiple studies has raised questions about the ability of endoscopy to reduce CRC mortality in the right colon.(46-48) Thus, it is unclear what the overall impact of endoscopy (e.g., colonoscopy screening) is, and whether there may be a large difference in impact on the left side of the colon compared with the right side.(46) The SCORE RCT from Italy randomly assigned 34,272 participants aged 55 to 64 years to either one-time FS or control. About 58% of FS group participants actually had an FS. After 10.5 years of follow-up, CRC incidence was reduced in the FS group by 18% (RR, 0.82; 95% CI, 0.69–0.96), with the reduction beginning about 5 years after randomization. CRC mortality in the FS group was also lower than in the control group, but not to a statistically significant degree (RR, 0.78; 95% CI, 0.56–1.08). Overall, these results are consistent with the United Kingdom results.(49) Sigmoidoscopy screening was also evaluated in the Prostate, Lung, Colorectal, and Ovarian Cancer (PLCO) screening randomized trial.(50) Results were very similar to those of the United Kingdom trial. In PLCO, 77,445 men and women aged 55 to 74 years were randomly assigned to receive 60 cm flexible sigmoidoscopy screening at baseline and again at 3 or 5 years from 1993 to 2001 depending on their date of entry into the trial. The 77,455 men and women randomly allocated to the control arm received usual medical care. An examination was considered positive if a polyp or mass was detected. Participants were then referred to their primary care physicians for diagnostic follow-up. A total of 86.6% of participants underwent at least one sigmoidoscopy screening and 28.5% were positive. Of these, 80.5% had a diagnostic evaluation, of which 95.6% underwent colonoscopy. Thus 21.9% of participants in the intervention arm had a colonoscopy.

Colorectal cancer screening in the control arm (contamination) was assessed by a questionnaire in a small sample of participants. The rate was estimated as 46.5%. In addition, the rate of colonoscopy after the screening phase was estimated as 47.7% in the intervention arm and 48.0% in the control arm.

The primary endpoint was colorectal cancer mortality, with colorectal cancer incidence a key secondary endpoint. All cancers and deaths were ascertained primarily by means of an annual questionnaire. Median follow-up time was 11.9 years and vital status within a year of the cutoff date was known for 99.9% of participants. The incidence rate of colorectal cancer was 11.9 per 10,000 person years in the intervention arm (1,012 cases) versus 15.2 in the usual care arm (1,287 cases) yielding a statistically significant RR of 0.79 (95% CI, 0.72–0.85). The absolute colorectal cancer risk reduction was 0.35%. In the distal colon the RR was 21

RIBOGRAMA PROJECT 0.71 (95% CI, 0.64–0.80) while in the proximal colon the RR was 0.86 (95% CI, 0.76–0.97). The colorectal cancer mortality rate was 2.9 deaths per 10,000 person-years in the intervention arm (252 deaths) versus 3.9 deaths in the usual care arm (341 deaths) for a statistically significant RR of 0.74 (95% CI, 0.63–0.87). The absolute reduction in risk of colorectal cancer death was 0.11%. The mortality RR for the distal colon was 0.50 (95% CI, 0.38–0.64) while that for the proximal colon was 0.97 (95% CI, 0.77–1.22). Treatment of diagnosed colorectal cancers was very similar by arm within each stage. The rate of bowel perforations was 2.8 per 100,000 sigmoidoscopies. False positive screening results were observed in 20% of men and 13% of women.

The RR for deaths from all causes excluding prostate, lung, colorectal, and ovarian cancers was 0.98 (95% CI, 0.96–1.01).(51) Two case-control studies have been reported that evaluate the efficacy of screening sigmoidoscopy in preventing CRC mortality;(51,52) one study used rigid sigmoidoscopy, and the other used rigid and FS. Both studies were conducted in prepaid health plans and suggested a significantly decreased risk (70%–90%) of fatal cancer of the distal colon or rectum among individuals with a history of one or more sigmoidoscopic examinations compared with nonscreened patients.

There are no strong direct data to determine frequency of screening tests in programs of screening.

3 - COMBINATION OF FOBT AND FLEXIBLE SIGMOIDOSCOPY A combination of FOBT and sigmoidoscopy might increase the detection of lesions in the left colon (compared with sigmoidoscopy alone) while also increasing the detection of lesions in the right colon. Sigmoidoscopy detects lesions in the left colon directly but detects lesions in the right colon only indirectly when a positive sigmoidoscopy (that may variously be defined as a finding of advanced adenoma, any adenoma, or any polyp) is used to trigger a colonoscopic examination of the whole colon.

In 2,885 veterans (97% male; mean age 63 years), the prevalence of advanced adenoma at colonoscopy was 10.6%. It was estimated that combined screening with one-time FOBT and sigmoidoscopy would detect 75.8% (95% CI, 71.0%–80.6%) of advanced neoplasms. Examination of the rectum and sigmoid colon during colonoscopy was defined as a surrogate for sigmoidoscopy. This represented a small but statistically insignificant increase in the rate of detection of advanced neoplasia when compared with FS alone (70.3%; 95% CI, 65.2%–75.4%). The latter result could be achieved assuming that all patients with an adenoma in the distal colon undergo complete colonoscopy. Advanced neoplasia was defined as a lesion measuring at least 10 mm in diameter, containing 25% or more villous histology, high-grade dysplasia, or invasive cancer.(53) A study of 21,794 asymptomatic persons (72% were men) who had both colonoscopy and fecal immunochemical testing (FIT) for occult blood compared the detection of right-sided cancers as triggered by different test results. FIT alone resulted in a sensitivity of 58.3% and a specificity of 94.5% for proximal cancer diagnosis. FIT plus the finding of advanced neoplasia in the rectosigmoid colon yielded a sensitivity of 62.5% and a specificity of 93%. Thus, in this trial, the addition of sigmoidoscopy to FIT did not substantially improve the detection of right-sided colon cancers, compared with FIT alone.8%.54) The NORCCAP once-only screening study randomly assigned 20,780 men and women, aged 50 to 64 years, to FS only or a combination of FS and FOBT with FlexSure OBT.(55) A positive FS was defined as a finding of any neoplasia or any polyp at least 10 mm. A positive FS or FOBT qualified for colonoscopy. Attendance in this study was 65%. Forty-one cases of CRC were detected (0.3% of screened individuals). Adenomas were found in 2,208 participants (17%), and 545 (4.2%) had high-risk adenomas. There was no difference in diagnosis yield between the FS only and the FS and FOBT groups regarding CRC or high-risk adenoma.

22

RIBOGRAMA PROJECT There were no serious complications after FS, but there were six perforations after therapeutic colonoscopy (1:336).

4 - BARIUM ENEMA As part of the National Polyp Study, colonoscopic examination and barium enema were compared in paired surveillance examinations in those who had undergone a prior colonoscopic polypectomy.(56) The proportion of examinations in which adenomatous polyps were detected by barium enema was related to the size of the adenoma (P = .009); the rate was 32% for colonoscopic examinations in which the largest adenomas detected were no larger than 5 mm, 53% for those in which the largest adenomas detected were 6 mm to 10 mm, and 48% for those in which the largest adenomas detected were larger than 10 mm. In patients who have undergone colonoscopic polypectomy, colonoscopic examination is a more sensitive method of surveillance than double-contrast barium enema.

5 - COLONOSCOPY Because there are no RCTs of colonoscopy, evidence of benefit is indirect. Most indirect evidence is about detection rate of lesions that may be clinically important (like early CRC or advanced adenomas). Some casecontrol results are available. One RCT of colonoscopy has been initiated.(57) EVIDENCE ABOUT LESION DETECTION RATE In a colonoscopic study of 3,121 predominantly male U.S. veterans (mean age: 63 years), advanced neoplasia (defined as an adenoma that was ≥10.0 mm in diameter, a villous adenoma, an adenoma with high-grade dysplasia, or invasive cancer) was identified in 10.5% of the individuals.(58) Among patients with no adenomas distal to the splenic flexure, 2.7% had advanced proximal neoplasia. Patients with large adenomas (≥10.0 mm) or small adenomas (<10.0 mm) in the distal colon were more likely to have advanced proximal neoplasia than were patients with no distal adenomas (OR = 3.4; 90% CI, 1.8–6.5 and OR = 2.6; 90% CI, 1.7– 4.1, respectively). One-half of those with advanced proximal neoplasia, however, had no distal adenomas. In a study of 1,994 adults (aged 50 years or older) who underwent colonoscopic screening as part of a program sponsored by an employer, 5.6% had advanced neoplasms.(59) Forty-six percent of those with advanced proximal neoplasms had no distal polyps (hyperplastic or adenomatous). If colonoscopic screening is performed only in patients with distal polyps, about half the cases of advanced proximal neoplasia will not be detected.

A study of colonoscopy in women compared the yield of sigmoidoscopy versus colonoscopy. Among 1,463 women, cancer was found in one woman and advanced colonic neoplasia in 72 women or 4.9% (about onehalf the prevalence compared with men). The authors focused, however, on RR (i.e., RR of missing an advanced neoplasm) as the outcome, instead of absolute risk of such neoplasms, which is substantially lower in women. In addition, the natural history of advanced neoplasia is not known, so its importance as an outcome in studies of detection is not clear.(60) Analysis of data from a colonoscopy-based screening program in Warsaw, Poland demonstrated higher rates of advanced neoplasia in men than in women. The predominant age range of participants was 50 to 66 years.

Of the 43,042 participants aged 50 to 66 years, advanced neoplasia was detected in 5.9% (5.7% among women with a family history of CRC, 4.3% among women without a family history of CRC, 12.2% among men with a family history of CRC, and 8.0% among men without a family history of CRC). Clinically significant complications requiring medical intervention were rare (0.1%) consisting of five perforations, 13 episodes of 23

RIBOGRAMA PROJECT bleeding, 22 cardiovascular events, and 11 other events over the entire population of 50,148 screened persons. There were no deaths; however, the author reported that collection of 30-day complications data was not systematic (therefore, the data may not be reliable).(61) Detection rates in colonoscopy screening vary with the rate at which the endoscopist examines the colon while withdrawing the scope. Detection rates among gastroenterologists (mean number of lesions per patient screened, 0.10â&#x20AC;&#x201C;1.05; range of the percentage of patients with adenomas, 9.4%â&#x20AC;&#x201C;23.7%) and the times to withdraw (3.1â&#x20AC;&#x201C;16.8 minutes for procedures not including polyp removal). Examiners whose mean withdrawal time was 6 minutes or more had higher detection rates than those with mean withdrawal times of less than 6 minutes (28.3% vs. 11.8%; P < .001 for any neoplasia) and (6.4% vs. 2.6%; P < .005 for advanced neoplasia).(62) Overall detection rate of adenomas and cancer may be affected by how thoroughly endoscopists search for flat adenomas and flat cancer. While the phenomenon of flat neoplasms has been appreciated for years in Japan, it has more recently been described in the United States. In a study in which endoscopists used high-resolution white-light endoscopes, flat or nonpolypoid lesions were found to account for only 11% of all superficial colon lesions, but they were about 9.8 times as likely to contain cancer (in situ neoplasia or invasive cancer) compared with polypoid lesions.(41) However, because the definition of flat or nonpolypoid was height less than one-half of the diameter, it is likely that many lesions classified as nonpolypoid in this study would be routinely found and described by U.S. endoscopists as sessile. At the same time, the existence of very flat or depressed lesions (depressed lesions are very uncommon but are highly likely to contain cancer) means that endoscopists will want to pay increasing attention to this problem.(63) Flat lesions may play a role in the phenomenon of missed cancers.(64) Flat or difficult-to-detect lesions include "serrated polyps," which may be more common in the right colon than they are in the left. The term serrated polyp is currently used to include hyperplastic polyps, sessile serrated adenomas, traditional serrated adenomas, and mixed serrated polyps.(65,66) The clinical significance of these lesions is uncertain, because the natural history is so difficult to learn for any polypoid lesion. However, the histologic and molecular characteristics of some serrated lesions suggest possibly important malignant potential (mutations in the BRAF gene may be an early step toward carcinogenesis in serrated polyps).(66) This potential, along with the challenges of detecting flat lesions, may partially account for recent reports of a colonoscopy's lesser protective effect in the right colon compared to the left.

In 2011, authors of one study reported variability of detection rates for proximal serrated polyps. They studied 15 colonoscopists on faculty at one university and showed, during the years 2000 to 2009, a wide variation in detection rate for proximal serrated polyps, ranging (per colonoscopy) from 0.01 to 0.26, suggesting that many proximal serrated lesions may be missed on routine exam.(65) The overall proportion of polyps that are "serrated" is unknown, in part because these lesions have been unappreciated and/or difficult to identify.

EVIDENCE ABOUT COLORECTAL CANCER MORTALITY REDUCTION Although there is no RCT to assess reduction of CRC incidence or mortality by colonoscopy, some casecontrol evidence is available. Based on case-control data about sigmoidoscopy, noted above, it has been speculated in the past that protection for the right colon might be similar to that found for the left colon.

However, a recent case-control study of colonoscopy raises questions about whether the impact of colonoscopy on right-sided lesions might be different than the impact on left-sided lesions.(47) Using a province-wide administrative data base in Ontario, investigators compared cases of persons who had received a diagnosis of CRC from 1996 to 2001 and had died by 2003. Controls were selected from persons who did not die of CRC. Billing claims were used to assess exposure to previous colonoscopy. The OR for the association between complete colonoscopy and left-sided lesions was 0.33, suggesting a substantial mortality reduction. For right-sided lesions, however, the OR of 0.99 indicated virtually no mortality reduction.

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RIBOGRAMA PROJECT This difference, which was striking and unexpected, might be explained in several possible ways and has been discussed extensively.(67) It is possible that exams were incomplete and did not reach the cecum even though they were coded as complete. It is possible that poor prep or incomplete mucosal inspection caused important lesions to be missed. It is also possible that examination was complete but some right-sided lesions simply grow rapidly and are not detected and treated by periodic colonoscopy. In other words, it is impossible to determine the reason for the dramatically different results on the right side compared to the left side, which are either the result of physician-related or patient-related factors or are the result of the biology of cancer.

Even if it is not possible to determine which reasons may be responsible for the right side versus the left side difference, the findings beg the question, “What is the degree of mortality reduction from colonoscopy?” While a figure of 90% is sometimes cited as the degree of mortality reduction,(68) the question will not be properly answered until the European study is completed.(57) In the meantime, the results of the study mentioned above (47) question what is known about the degree of CRC mortality reduction provided by colonoscopy.

Until there are more reliable results from RCTs, it may be prudent to expect an approximately 60% to 70% CRC mortality reduction, based on consideration of these studies and other data.(67) EVIDENCE ABOUT COLORECTAL CANCER REDUCTION A case-control study assessed CRC reduction (not CRC mortality reduction) in the right side versus the left side. In a population-based study from Germany, data were obtained from administrative records and medical records; 1,688 case patients (with CRC) were compared with 1,932 participants (without CRC), aged 50 years or older.(46) Data were collected about demographics, risk factors, and previous screening examinations.

According to colonoscopy records, the cecum was reached 91% of the time. Colonoscopy in the previous 10 years was associated with an OR for any CRC of 0.23, for right-sided CRC of 0.44, and for left-sided CRC of 0.16. While this study does not assess CRC mortality, the results suggest that the magnitude of the right side versus the left side difference may be smaller than previously found.(47) It would be extremely useful to assess right side versus left side differences in a RCT.

6 - VIRTUAL COLONOSCOPY (COMPUTED TOMOGRAPHIC [CT] COLONOGRAPHY) Virtual colonoscopy (also known as CT colonography or CT pneumocolon) refers to the examination of computer-generated images of the colon constructed from data obtained from an abdominal CT examination.

These images simulate the effect of a conventional colonoscopy. Patients must take laxatives to clean the colon before the procedure, and the colon is insufflated with air (sometimes carbon dioxide) by insertion of a rectal tube just prior to radiographic examination.(69) A large, paired-design study was conducted by the American College of Radiology Imaging Network group, with 2,531 average-risk people (prevalence of polyps or cancer greater than or equal to 10 mm = 4%; mean age about 58 years) screened with both CT colonography and optical colonoscopy (OC). The gold standard was the OC, including repeat OC exams for people with lesions found by computed tomographic colonography (CTC) but not by OC. Of 109 people with at least one adenoma or cancer greater than or equal to 10 mm, 98 (90%) were detected by CTC (referring everyone with a CTC lesion of 5 mm or greater). Specificity was 86% and PPV was 23%. There are several concerns from this study, including the following:  

Most, but not all, lesions found by CTC and not by OC were followed up with repeat OC. The design itself did not allow for following patients, thus potentially missing lesions that grow rapidly and would only be seen after follow-up.

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Because the centers conducting the screening were primarily academic centers and the radiologists and endoscopists were carefully trained, the generalizability of the findings is not clear.

Sixteen percent of people had an extracolonic finding that required further evaluation.

Unknowns from the study include the following for either OC or CTC: the number of detected polyps that would have progressed to invasive cancer, and the number of people harmed by the screening process.(70)Another study reported similar sensitivity and specificity in persons with an increased risk of CRC.(71) In this study, the sensitivity of OC could not be determined because it was done in an unblinded manner. This study suggests that virtual colonoscopy might be an acceptable screening or surveillance test for persons with a high risk of CRC, but this cross-sectional study does not address outcome or frequency of testing in high-risk persons.

Some studies have assessed how well virtual colonoscopy can detect colorectal polyps without a laxative prep. The question is of great importance for implementation because the laxative prep required by both conventional colonoscopy and virtual colonoscopy is considered a great disadvantage by patients. By tagging feces with iodinated contrast material ingested during several days prior to the procedure, investigators in one study were able to detect lesions larger than 8 mm with 95% sensitivity and 92% specificity.(72) The particular tagging material used in this study caused about 10% of patients to become nauseated; however, other materials are being assessed.

Another study (73) utilized low fiber diet, orally ingested contrast, and 'electronic cleansing', a process that subtracts tagged feces. CTC identified 91% of persons with adenomas 10 mm or larger, but detected fewer (70%) lesions greater than or equal to 8 mm. Patients who received both CTC and optical colonoscopy preferred CTC to optical colonoscopy (290 vs. 175). This study shows that CTC without a laxative prep detects small 1 cm lesions with high sensitivity and is acceptable to patients. Long-term utilization of CTC will depend on several issues including the frequency of follow-up exams that would be needed to detect smaller lesions that were undetected and may grow over time.

Extracolonic abnormalities are common in CT colonography. Fifteen percent of patients in an Australian series of 100 patients, referred for colonography because of symptoms or family history, were found to have extracolonic findings, and 11% of the patients needed further medical workups for renal, splenic, uterine, liver, and gallbladder abnormalities.(74) In another study, 59% of 111 symptomatic patients referred for clinical colonoscopy in a Swedish hospital between June 1998 and September 1999 were found to have moderate or major extracolonic conditions on CT colonography. CT colonography was performed immediately prior to colonoscopy and these findings required further evaluation. It is unstated to what extent the follow-up of these incidental findings benefited patients.(75) Sixty-nine percent of 681 asymptomatic patients in Minnesota had extracolonic findings, of which 10% were considered to be highly important by the investigators, requiring further medical workup. Suspected abnormalities involved kidney (34), chest (22), liver (8), ovary (6), renal or splenic arteries (4), retroperitoneum (3), and pancreas (1);(76) however, the extent to which these findings will contribute to benefits or harms is uncertain. Two other studies, one large (n = 2,195) and one small (n = 136) examined the moderate or high importance of extracolonic findings from CTC. The larger study (77) found that 8.6% of patients had an extracolonic finding of at least moderate importance, while 24% of patients in the smaller study (78) required some evaluation for an extracolonic finding. The larger study found nine cancers from these evaluations, at a partial cost (they did not include all costs) of $98.56 per patient initially screened. The smaller study found no important lesions from evaluation, at a cost of $248 per person screened. Both of these estimates of cost are higher than previous studies have found. The extent to which any patients benefited from the detection of extracolonic findings is not clear. Because both of these studies were conducted in academic medical centers, the generalizability to other settings is also not clear. Neither of these studies examined the effect of extracolonic findings on patient anxiety and psychological function.

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Technical improvements involving both the interpretation methodology, such as three dimensional (3-D) imaging, and bowel preparation are under study in many centers. While specificity for detection of polyps is homogeneously high in many studies, sensitivity can vary widely. These variations are attributable to a number of factors including characteristics of the CT scanner and detector, width of collimation, mode of imaging (two dimensional [2-D] vs. 3-D and/or "fly-through"), and variability in expertise of radiologists.(79) DIGITAL RECTAL EXAMINATION A case-control study reported that routine digital rectal examination was not associated with any statistically significant reduction in mortality from distal rectal cancer.(80)

7 - DETECTION OF DNA MUTATIONS IN THE STOOL The molecular genetic changes that are associated with the development of colorectal adenomas and carcinoma have been well characterized.(81) Advanced techniques have been developed to detect several of these gene mutations that have been shed into the stool.(82-85) Stool DNA testing was recently assessed in a prospective study of asymptomatic persons who received colonoscopy, three-card FOBT (Hemoccult II), and stool DNA testing based on a panel of markers assessing 21 mutations. Conducted in a blinded way with prestated hypotheses and analyses, the study found that among 4,404 patients, the DNA panel had a sensitivity for CRC of 51.6% (for all stages of CRC) versus 12.9% for Hemoccult II, while the false-positive rates were 5.6% and 4.8%, respectively. On this basis, the approach looks promising but would be improved, if possible, by increased sensitivity (perhaps by increasing the number of DNA markers) and by reduced cost.(86,87)

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66. Hoff G, Grotmol T, Skovlund E, et al.: Risk of colorectal cancer seven years after flexible sigmoidoscopy screening: randomised controlled trial. BMJ 338: b1846, 2009 67. Thiis-Evensen E, Hoff GS, Sauar J, et al.: Population-based surveillance by colonoscopy: effect on the incidence of colorectal cancer. Telemark Polyp Study I. Scand J Gastroenterol 34 (4): 414-20, 1999.

68. Atkin WS, Edwards R, Kralj-Hans I, et al.: Once-only flexible sigmoidoscopy screening in prevention of colorectal cancer: a multicentre randomised controlled trial. Lancet 375 (9726): 1624-33, 2010.

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70. Brenner H, Chang-Claude J, Seiler CM, et al.: Protection from colorectal cancer after colonoscopy: a population-based, case-control study. Ann Intern Med 154 (1): 22-30, 2011.

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92. Smith MJ: Colon cancer screening hitting its stride but obstacles impairs reaching final goal.

Gastroenterology and Endoscopy News 59 (3): 9, 2008. [Refer to "Screening: it’s not just a good idea, it’s the law … almost" within the article].

93. Ferrucci JT: Colon cancer screening with virtual colonoscopy: promise, polyps, politics. AJR Am J Roentgenol 177 (5): 975-88, 2001.

94. Johnson CD, Chen MH, Toledano AY, et al.: Accuracy of CT colonography for detection of large adenomas and cancers. N Engl J Med 359 (12): 1207-17, 2008.

95. Regge D, Laudi C, Galatola G, et al.: Diagnostic accuracy of computed tomographic colonography for the detection of advanced neoplasia in individuals at increased risk of colorectal cancer. JAMA 301 (23): 2453-61, 2009.

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97. Zalis ME, Blake MA, Cai W, et al.: Diagnostic accuracy of laxative-free computed tomographic colonography for detection of adenomatous polyps in asymptomatic adults: a prospective evaluation.

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V - CRC EPIDEMIOLOGY AND RESOURCE ALLOCATION Most OECD countries have implemented health care reform as a means of improving system-wide efficiency and attempting to control escalating health care costs. Despite a series of incremental changes in health system organization, the general trend towards decentralization of the purchasing agent has been an unmistakable phenomenon. In many instances the strategic movement of the purchasing function to more localized settings, has been used to consolidate a heightened sense of financial accountability and encourage local autonomy. Such changes in health-system organization have important implications with regard to the local purchaser ability to cater to specific population health need. Given that such arrangements have a tendency to encourage risk segmentation, resource allocation mechanisms are important when responding to population need.

In this context, this chapter examines how disease burden across Europe and Australia guides resource allocation mechanisms. The chapter focuses primarily on how CRC influences resource allocation mechanisms across the study countries and is split into two main sections: the first, assesses the current burden of CRC across Europe and Australia, introduces CRC epidemiological trends, primarily around traditional indicators such as incidence, mortality and survival, and examines variations in disease burden over the last decade. The second section examines how resources are allocated at various stages of the CRC management pathway, assesses the current context for CRC funding, by identifying explicit forms of diseaserelated resource allocation, and examines how various aspects of management influence funding mechanisms.

1 - CRC INCIDENCE AND MORTALITY Colorectal cancer (CRC) accounts for significant morbidity and mortality across Europe and Australia. Indeed, after breast cancer (ASR=94.31), CRC (ASR=55.4) is the highest diagnosed cancer in women and the third most amongst males, after prostate (ASR=86.7) and lung (ASR=75.3) cancer. As a proportion of European cancer incidence, Age Standardised Rate measuring number of cases per 100,000 population.

CRC incidence accounted for 12% and 14% of total cancers in Europe in 1995, and 12.3% and 13.1% in 2006 in males and females respectively. The greatest cause of cancer mortality throughout Europe in men is lung cancer (ASR=64.8), followed by CRC (ASR=27.3) and prostate (ASR=22.2) cancers. In women the primary cause for cancer mortality is breast cancer (ASR=26.0), then CRC (ASR=16.6) and lung (ASR=15.1) cancers.

CRC mortality as a percent of total cancers in Europe was 10% and 14% in 1995, and both 12.2% in 2006 for males and females respectively.3 In many European countries incidence has increased since 1995 while mortality has fallen (mortality ratio of 1.8 in 1995 and 2.0 in 2006). There are noted differences between European countries for incidence and mortality. Ultimately, it appears both Northern and Eastern Europe have higher CRC incidence and mortality than Southern Europe, most markedly Greece and Albania. There have been a number of hypotheses for this pattern.

It may reflect greater fruit, vegetable, fiber and fish consumption and more active lifestyles in the south than elsewhere; while poorer prognosis in the east may reflect poorer treatment and advanced stage at diagnosis.

There may also be genetic differences across Europe thereby creating lower proportions of high risk individuals in the south than north. The increase in CRC incidence is likely to continue with aging populations: by 2015 the population of >65 years will have increased by 22% compared to 2000, and the population of >80 32

RIBOGRAMA PROJECT years will increase 50%.5 The pattern of age related CRC incidence, and lesser so mortality, is closely correlated with increasing age from around 50 years, although the proportions differ between countries.

Both incidence and mortality appear to be greater in men than in women; comparison of 1995 and 2006 cohorts found greater rises in incidence (>5% absolute difference) in men than women. There may be a number of factors accounting for this gender difference. There may be inherent biological differences in women providing added protection against CRC, such as hormonal or genetic factors. There may also be lifestyle difference between the sexes such as greater fruit and vegetable consumption in women than men, a hypothesis supported by the finding of greater plasma carotenoids in women than men in across Europe.

Sources: Bray F, Sankila R, Ferlay J et al. 2002; and Ferlay J, Autier P, Boniol M et al.2007.

2 - CRC SURVIVAL Survival of patients with CRC differs markedly across countries. In comparison with other cancers, CRC survival fares marginally better than average (EUROCARE-4: CRC 53.7% 5 year relative survival versus all cancers 51.9%). When examined in comparison with other cancers with high incidence in Europe, CRC does not fare 30 as well. In comparison the latest European 5 year relative survival for breast cancer is 81.1%, for prostate cancer 77%, although for lung cancer survival is poor at 12.6%. The recently published EUROCARE-4 survey has not published sex-specific survival figures, but previous EUROCARE studies have found sex-specific survival differences. In the majority of countries survival is better for women than men. In contrast, the American SEER data report a range of 60-65% for 5 year survival between men and women. Again there appears to be regional differences in survival, with poor CRC survival particularly in Eastern Europe. Previous EUROCARE surveys also found poor survival in some Northern European countries, most notably the UK and Denmark. The latest data, however, no longer supports this trend for all Northern European countries, except for the UK and Denmark. Europe overall has improved survival by 4.2 percentage points, and certain countries have increased between 5 and 10 points (Czech Republic, Germany, Italy, Poland, Slovenia, UK). Indeed, most countries now meet moderate prognosis (40-59%) for CRC survival, except for Poland.

There are a number of hypotheses to explain these differences between countries.

Early detection appears to be a significant predictor for survival, and correcting for stage at diagnosis shrinks country differences somewhat.9 Since the EUROCARE surveys began, detection of CRC has improved and (pilot) population screening programs have begun in select European countries. Additional factors affecting survival between countries are proportion of adenocarcinomas in polyps (less are associated with improved prognosis), higher proportion of tumours not microscopically verified, and access to curative surgery.

Furthermore, treatment for CRC has also improved with the advent of better surgery, radiotherapy and combination chemotherapy.

Source: Berrino F, et al, 2007.

France has been consistently one of the better performing countries for CRC survival. In contrast, the UK is one of the poorer performing countries with its recent EUROCARE result ranking 10th out 14 (many countries share the same ranking). Only Denmark fares worse for Northern and Southern European countries. This ranking has not improved with time, although there have been marked improvements since survival data begun collecting. Survival of curative resection in all stages has increased since 1974 in the UK, due to downstaging of the disease (earlier detection), improved diagnostic staging, and improvements in treatment and peri-operative care.

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VI - CRC RESOURCE ALLOCATION IN EUROPE AND AUSTRALIA The use of formal resource allocation across Europe is very patchy. Use of formal resource allocation mechanisms was reported in the Czech Republic, Romania, Greece, Italy, the Netherlands, Spain, the UK and Australia. Furthermore mechanisms of formal resource allocation were mainly seen to be prospective. More specifically, countries tended to allocate a fixed amount of resources to ‘purchasers’ based on expected future expenditure. In the UK, semi-autonomous primary care trusts (PCTs) were allocated funds based on a set of proxy indicators used to define local population need. These were based on a series of demographic and socio-economic indicators, in addition to the current status disease burden at the local level. On the other hand in the Australian public healthcare-system (which uses a publicprivate financing) money was allocated from the federal to the state governments on the basis of a fiveyear Australian Healthcare Agreement.18 Within this arrangement, the federal government allocates a five year lump sum to state governments, ex-ante. The money is then used to fund public hospital health services within the area. The only exception to this general trend of prospective budgeting was the Netherlands, where, although use of ‘formal’ resource allocation was reported, fund distribution took the form of risk equalization rather than the distribution of funds from the source of pooling to the purchaser. Countries that used social health insurance as a main method of financing healthcare were found to lack formal methods of resource allocation. Use of social health funds as the main collector of revenue, the locus of financial pooling as well as the purchaser of care, meant that there was an acute lack of financial distribution in the form of prospective budgeting in western and central Eastern Europe. Absence of ‘formal’ resource allocation was reported in Denmark, Germany, France, Hungary, Portugal, the Russian Federation, Slovakia, Sweden and Turkey. Out of the nine countries that reported no formal resource allocation, five used social health insurance systems as the main method of finance (France, Hungary, Turkey, Slovakia and Germany). In all instances, the main form of financial allocation took place between the purchaser of care and provider of care. In Germany, statutory care in the form of medical services was reimbursed using a DRG payment system. In addition, in Hungary a system of payment based on the American DRGs and the German fee-for-service points system was used to finance out and inpatient care. In all of these countries, central government played a critical role in sanctioning the reimbursement of services and thus coordinating the rationing of care.

Healy, Sharman and Lonkuge, 2006 indicate that the Ministry of Health plays a significant role in the coordination of expenditure. In Germany this occurred by way of the Federal Joint Committee. Placed at a national level, this organization has the responsibility of defining the scope of reimbursement within ambulatory care. Furthermore within countries that described resource allocation as being ‘informal’, a more interesting phenomenon presented itself. Despite informal resource allocation characterized by retrospective payment type, certain forms of prospective budget allocation were also seen as informal. In these settings budgets were based on various transitory arrangements. A prime example of this was seen in Russia with the ‘left-over’ principle. The Russian health care system is based on many sources of revenue that are collected from federal, regional and local taxes, as well as social health insurance contributions. In the Russian case, funds from specific revenue sources are designated to specific hospital services, and therefore play an essential part framing patient care. However, in this setting, there is no clear and transparent mechanism for defining how much money should be allocated to which services. This creates problems as to which services should be provided for free and which should be paid for out of pocket. The use of a complex allocation mechanism was a clear phenomenon with the majority of European countries and Australia which used a hybrid system of allocation to distribute funds at the national, regional and local level. These levels of distribution were found to arise from historical issues surrounding regional autonomy or, in some cases, functions of recently 34

RIBOGRAMA PROJECT implemented health sector reforms. This was observed particularly in Denmark, Italy, Romania, Spain, the UK and Australia. Given the significant degree of regional autonomy in Spain and Italy, resource allocation from the federal to the state level played a significant part in determining region-specific budgets. In Denmark, on the other hand, revenue collection and the subsequent pooling of funds occurring at the regional level has caused there to be a series of risk equalization mechanisms, to avoid risk segmentation.

Essentially taxes levied at the national level are reallocated based on risk. However in the UK, the relatively recent split between the purchaser and provider has lead to a series of interesting allocation processes. At this moment in time the UK government allocates an estimated 80% of the national health budget to PCTs. Although funds are simultaneously allocated to the NHS Hospital Trusts, the bulk of resource distribution occurs at the local level; in the form of PCT ‘payment’ for services conducted within the hospital setting. In the majority of cases, resource mobilization between the two organization is based on a complex array of globalised budgets (defined by historical analysis of past use), service lead agreements (based on cost and value agreements made between the two parties) and targeted payment by results (PbR).

Finally, risk equalization mechanisms, in their own right, seemed to play a prominent part in the overall amount of money allocated to purchasers of care. This was seen mostly within countries that presented social health insurance schemes as the main mode of healthcare finance. Indeed, this was the main way that such countries tried to safeguard against risk-segmentation. A prime example of this was seen within the Netherlands where the purchasers of care were seen to adjust budgets based on the risk profile of the specified population. Formulas used for reallocation were based on age, sex, socioeconomic deprivation, sources of income, and socioeconomic status. Equalization formulas also took into account measures of disease burden. This was seen in the form the creation of specific cost-groups for expensive drugs and DRG coding for chronic disease. These indicators were used to distribute funds accordingly; at the beginning and end of the year. Allocation specifically to cancer was found to be extremely limited, with disease specific allocation only in Denmark, the Russian Federation, Romania, Australia and the UK. Across the 18 countries studied, Australia was the only country to report the use of the explicit allocation process for all cancers. Denmark, the Netherlands, Romania and the UK only reported explicit mechanisms of resource allocation to cancer. Yet closer analysis of these methods of resource distribution found significant differences with regard to the distribution of funds. In Romania, funds available from the National Cancer Program were allocated to District Health Insurance Authorities on a monthly basis. In the UK money allocated to cancer was given on a yearly basis. However, in the Russian Federation oncology care is not covered by medical insurance and is exclusively funded by federal and regional budgets. Given that the majority of care is given in specialist institutions, in most cases resources were allocated on a per diem basis, with some additional cost for the consultation time. These arrangements created significant issues relating to underfunding. Particular forms of treatment tended to be financed using cost estimations. However, given that these values were defined without transparent and clear mechanisms, the costs of treatment in real terms often exceeded proposed estimates.

Many respondents indicated that the resources allocated to cancer in general and CRC in particular, were insufficient, although in the majority of cases no appropriate measure of need versus expenditure was available to substantiate this claim, but gaps in specific parts of the care delivery pattern were mentioned. Of the study countries, only the UK, Greece and the Netherlands indicated that the resources allocated to cancer and CRC care were ‘sufficient’.

While countries such as the UK cited the recent levels of increased investment in cancer overall as the reasoning behind this opinion, the majority of countries highlighted lack of facilities, shortage in the clinical skill-set and poor access to pharmaceuticals, as the reasoning behind this negative response. More specifically, delays in the inclusion of important novel drugs on the Hungarian reimbursement list placed inherent barriers to pharmaceutical access. Furthermore, long diagnosis and treatment waiting times, 35

RIBOGRAMA PROJECT shortages of specialized physicians and overall lack of equipment was taken to be a sign of insufficient funding within the Portuguese and Russian cancer care program.

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VII - CANCER AND COLORECTAL CANCER SPENDING Only half of the surveyed countries seem to collect and report cancer expenditure and colorectal cancer expenditure, for most countries only on a periodic basis. In countries with poorer cancer registry data collection quality (Hungary, Slovakia, Russia) these numbers may be less accurate, however, for the remaining countries this adjustment helps estimate the cancer spending per cancer patient. The total cancer spending per total cancer incidence and mortality shows large variations between countries. The highest spending countries appear to be France, Germany and Sweden, while the lowest spending countries appear to be Russia and the Netherlands. For colorectal cancer spending adjusted for colorectal cancer burden, the highest spending countries appear to be France, Germany and the Netherlands, while the lowest appear to be Hungary and Spain.

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VIII - RESOURCE ALLOCATION FOR SCREENING Although all countries have some form of pilot CRC screening, the use of national CRC screening programmes is still quite limited. Furthermore, programmes within these countries were found to be at different stages of development. National CRC screening programmes exist in the Czech Republic, France, Italy Poland, Slovakia, the UK and Australia. In terms of years in operation, Slovakia presented one of the more established programmes, with CRC screening being part of a legislated reimbursement package since 2002. Under Act No. 577 of 2004 each person over 50 was able to receive CRC screening. In the UK, however, the national screening programme is still in the early stages of implementation, with CRC screening roll-out currently taking place between the years of 2006 and 2008. The majority of the remaining countries were still within the pilot stage (Denmark, Germany, Hungary the Netherlands, Romania, Spain and Sweden). Only Greece, Poland, the Russian Federation and Turkey, reported an absence of any form of pilot CRC screening.

Unlike CRC, the existence of national breast cancer screening programmes seems to be more widespread.

National programmes are running in the Czech Republic, Denmark, France, Germany, Hungary, Italy the Netherlands, Poland, Romania, the Russian Federation, Slovakia, Spain, the UK and Australia, and pilot programmes exist in Turkey. Greece and Portugal were the only countries to have no national or pilot breast screening programme.

Like CRC, the presence of national cervical screening was is also quite rare, with national programmes in the Netherlands, Poland, Romania, Slovakia, Sweden and the UK. Finally, Slovakia and Poland are the only countries in which national programmes for prostate screening are run.

Explicit allocation of resources to adult screening seemed to be standard within countries offering such programmes. Of countries that participated in some form of screening, explicit funding allocation was noted in Australia, France, Netherlands, Poland, Romania and the UK. Concrete values were only reported for six countries. In the Netherlands, spending for the 2005 IKA pilot was reported to be €700,000; this was based on a population of 32,000. CRC screening expenditure was €4.2 million in Poland (2005- 2006), while the pilot programme in Romania was allocated €185,000. Finally, although explicit CRC resource allocation was reported in Germany and the Czech Republic, no information was available regarding funding amounts.

With the exception of France and the UK, there still seems to be limited insight into the allocation methods used to finance screening initiatives. In France, programs are financed by the state, the national insurance scheme and the departments. Prior to 2004, financing of screening programs was compulsory for the French departments. Despite the previous screening legislation, roughly half of the departments continue to finance these programmes; utilizing a 13% share of funds in 2006. On the other hand in the UK, due to the fact CRC screening was still within the Phase 1 rollout funds were held centrally within the NHS Screening Plan. Funds were then allocated to screening hubs, using demographic indicators.

This calculation was based on the population composition of women, men and children. In this setting, screening hubs can use funds to respond to local health need. However such autonomy took place within the constraints of the screening protocol guideline. Eventually, it is hoped that PCTs will act as the purchaser of care, once the program has been nationalized. However it still unclear as to whether their will be a tariff, once screening is extended.

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RIBOGRAMA PROJECT The majority of countries in the survey did not report any amounts allocated and/or expended on breast cancer screening programmes. Only four countries reported this information. France, The Netherlands, Romania and the UK spent €31.5 million (2005), €40.7 million (2005), €700,000 (2006), and €107 million (2004), respectively. Limited information was available for other screening programmes. The Netherlands and Romania were the only countries to report a process in which explicit amounts of were given for cervical cancer screening programme. Specifically, the Netherlands allocated €26.1 million and Romania allocated €1.4 million.

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IX - CRC RESOURCE ALLOCATION TO DIAGNOSIS AND TREATMENT PROGRAMMES Denmark, France, Hungary, Poland, Romania, the Russian Federation, Slovakia, Sweden, the UK and Australia all had formal national management programmes for cancer. However, a considerable number of countries reported no such programmes. In Czech Republic, Germany, Greece, the Netherlands, Portugal, Spain and Turkey no formal cancer management programmes were present. Patterns on the availability of formal CRC diagnosis and treatment programmes across countries were also polarized. Formal CRC programmes were found in the Czech Republic, Denmark, Italy, the Netherlands, Poland, the Russian Federation, Sweden and the UK. However and perhaps surprisingly a lack of these programmes was found in Germany, Greece, Hungary, Portugal, Romania, Slovakia, Spain and Turkey. From the survey responses it is clear that a significant number of countries allocate funds specifically to cancer management programmes.

Such accommodations were made in Denmark, France, Hungary, Italy, Poland, Romania, Sweden, the UK and Australia.

Furthermore, it would seem, a small proportion of countries have a significant history of this activity. In 1991, Sweden allocated €623 million to cancer diagnosis and treatment programmes, while Denmark allocated more than €215 million, in the 1998-2003 period. In the UK more recent attempts to prioritize cancer diagnosis and treatment have led to the introduction of the National Cancer Plan (2000) and the Cancer Reform Strategy (2007), and these have led to an increased investment in cancer services. In 2006, some €6.24 million was spent on such programmes. Furthermore, Australia dedicated €117.4 million for the “Strengthening Cancer Care” initiative, over a five-year period. This initiative was introduced as part of the Australian government’s attempt to fulfill its election commitment to reduce the burden of cancer. Given that CRC presents a significant burden of disease in the majority of the study countries, there still seem to be an acute absence of targeted diagnosis and treatment plans.

Instead, the majority of countries studied tended to focus their attention on general cancer diagnosis and treatment programmes. Survey responses indicate that CRC diagnosis and treatment initiatives are present in only 7 of the countries studied (Denmark, the Czech Republic, Italy, the Netherlands, Poland, the Russian Federation, Sweden and the UK), while Germany, Greece, Hungary, Romania, Spain, Turkey and Australia reported no such programme. On the other hand, diagnosis and treatment programs for cancer overall, were used in the UK, Australia, Sweden, Slovakia, the Russian Federation, Romania, Poland, Italy, Hungary, France and Denmark.

The majority of the study countries had a serious lack of intelligence around the funding of national management programmes for CRC. The survey found data for the UK and the Netherlands only. However, even in these instances, UK data for such expenditure was not explicitly related to CRC diagnosis and treatment. In the second quarter of 2007, hospitals in the UK spent an average of €565,884 per 100,000 on all procedures based within the lower gastrointestinal canal. The Netherlands on the other hand reported CRC programme expenditure of €121,200.21

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X - RESUME Although the current burden of CRC is a significant and growing burden to health systems in developed countries, resource allocation systems have yet to make appropriate accommodations for it. Given that this â&#x20AC;&#x2DC;potentially avoidableâ&#x20AC;&#x2122; cancer is the second greatest cause of cancer death it is essential that funds are allocated in a way that is seen to be most effective. Indeed, the quest to lower CRC incidence through prevention initiatives, decrease rates of CRC mortality while improving CRC survival through early diagnosis and effective treatment, should be a hypothecated priority. However, the problem in achieving this goal is that for the majority of countries in this survey resource allocation mechanisms are not geared to explicitly allocate funds to individual cancers. In the majority of cases, population need is defined by socioeconomic, demographic and large composite indicators of general morbidity. This is certainly the case for taxbased systems and SHI-systems.

The only countries, within the study, to allocate hypothecated funding to CRC management programmes were Australia and the Netherlands. Yet even in these cases lessons taken from the theoretical principles of financing healthcare, indicate that there are a series of possible trade-offs that each system may have to contend with. For example, in the case of Australia, resource allocation in the form of budgets may increase financial accountability and allow policy analysts to monitor programme delivery and efficiency; however, this may hinder the resource flexibility needed to respond to changing needs of the population. Such arrangements may create budgeting silos or cause allocation of resources to be based on affordability rather than costeffectiveness. In the case of the Netherlands, retrospective risk equalization across SHI funds using DRG costing codes may represent an effective way of constructing and responding to population need. In contrast, however, this arrangement has in some cases been seen to bolster the incentive practices of up coding.

Furthermore, many countries lack the financial incentive structures needed to reinforce measures of diagnosis and treatment. Primarily, this stems from an acute lack of CRC-specific national management programmes in which care is delivered. Only eight of the study countries reported the existence of formal CRC diagnosis and treatment programmes. The majority of the treatment programs were organized around a general cancer plan.

Yet even in these circumstances, information relating to the specific amounts allocated to the general cancer plan was rare. The exceptions being Denmark and Sweden.

In sum, while countries have tried to respond to the current and future burden of CRC, existing resource allocation mechanisms within respective healthcare systems make it difficult for funds to be allocated in such a way that reflects the population need. Having said this, however, some countries have made the attempt to allocate funds based on specific cancers. Finally, lack of formal CRC national management programmes limit the potential to incentivize methods of best practice.

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References: 1. Al-Delaimy WK, van Kappel AL, Ferrari P et al. Plasma levels of six carotenoids in nine European countries: report from the European Prospective Investigation into Cancer and Nutrition (EPIC). Public Health Nutr 2004;7:713-22.

2. Australian Department of Health and Ageing. 2007. ‘Australian Health Care Agreements’: http://www.health.gov.au/internet/wcms/publishing.nsf/Content/health-ahca-agreement.htm 3. Australian Department of Health and Ageing. 2005. ‘Health Fact Sheet - Investing in Australia’s health: Strengthening Cancer Care’: http://www.health.gov.au/internet/budget/publishing.nsf/Content/healthbudget2005-Hbudgethfact1.htm.

4. Australian Government. Australian Institute of Health and Welfare. 2004. Most Frequently Occurring Cancer, 2004’: http://www.aihw.gov.au/cancer/data/excel_tables/most_common_cancers.xls 5. Berrino F, De Angelis R, Sant M et al. Survival for eight major cancers and all cancers combined for European adults diagnosed in 1995099: results of the EUROCARE-4 study. Lancet 2007 DOI:10.1016/S1470-2045(07)70245-0.

6. Bray F, Sankila R, Ferlay J et al. Estimates of cancer incidence and mortality in Europe in 1995. Eur J Cancer 2002;38:99-166.

7. Brenner H, Hoffmeister M, Arnkt V et al. Gender differences in colorectal cancer: implications for age at initiation of screening. Br J Cancer 2007; 96:828-31.

8. Bouhier, K., J. Maurel, et al. (2004). "Changing practices for diagnosis and treatment of colorectal cancer in Calvados: 1990-1999." Gastroenterol Clin Biol 28(4): 371-6.

9. Capocaccia R, Gatta G, Roazzi P, Carrani E, Santaquilani M, De Angelis R, Tavilla A, and the EUROCARE Working Group. The EUROCARE-3 database: methodology of data collection, standardization, quality control and statistical analysis. Annals of Oncology 2003; 14:v14-27 10. Centre for Genetics Education: http://www.genetics.com.au/services/canclin.html 11. Ciccolallo L, Capocaccia R, Coleman MP et al. Survival differences between European and US patients with colorectal cancer: role of stage at diagnosis. Gut 2005; 54:268-73.

12. Coleman MP, Gatta G, Verdecchia A et al. EUROCARE-3 summary: cancer survival in Europe at the end of the 20th century. Annals Oncol 2003; 14:v128-49.

13. Department of Health.

2007.

‘About NHS Allocations’: http://www.dh.gov.uk/en/Managingyourorganisation/Financeandplanning/Allocations /DH_076547 14. Department of Health. 2007. ‘Allocation of resources to English areas (AREA)': http://www.dh.gov.uk/en/Policyandguidance/Organisationpolicy/Financeandplanning/Allocations/DH _076547.

15. Engholm G, Kejs AMT, Brewster DH et al. Colorectal cancer survival in the Nordic countries and the United Kingdom: Excess mortality risk analysis of the 5 year relative period survival in the period 1999 to 2000. Int J Cancer 2007; 121:1115-22.

16. Faivre-Finn C, Bouvier-Benhamiche AM, Phelip JM et al. Colon cancer in France: evidence for improvement in management and survival. Gut 2002;51:60-64.

17. Ferlay J, Autier P, Boniol M et al. Estimates of the cancer incidence and mortality in Europe in 2006.

18. Annals Oncol 2007; doi:10.1093/annonc/md l498 Gastroenterology department, IPOFG Lisbon.

19. Gatta G, Ciccolallo L, Capocaccia R et al. Differences in colorectal cancer survival between European and US 20. populations: the importance of sub-site and morphology. Eur J Cancer 2003;39:2214-22.

21. Gatta G, Capocaccia R, Sant M et al. Understanding variations in survival for colorectal cancer in Europe: a EUROCARE high resolution study. Gut 2000;47;533-38.

22. Gatta G, Faivre J, Capocaccia R et al. Survival of colorectal cancer patients in Europe during the period 1978- 1989. Eur J Cancer 1998;34:2176-83.

23. Healy, Sharman and Lonkuge, 2006 42

RIBOGRAMA PROJECT 24. Hole DJ, McArdle CS. Impact of socioeconomic deprivation on outcome after surgery for colorectal cancer. Br J Surg 2002; 89:586-90.

25. Institut National du Cancer. 2006. Référentiels de Bon Usage hors GHS: http://www.ecancer.fr/v1/fichiers/public/rbu_digestif_6_160407.pdf 26. International Database. (2008). Mid-Year Population Statistics International Programmes Centre, United States Census Bureau, Population Division.

27. McArdle CS, McKee RF, Finlay IG et al. Improvement in survival following surgery for colorectal cancer. Br J Surg 2005; 92:1008-13.

28. Mitry E, Bouvier AM, Esteve J et al. Improvement in colorectal cancer survival: a population based study. Eur J Cancer 2005;41:2297-2303.

29. Mitry E, Bouvier AM, Esteve J et al. Benefit of operative mortality reduction on colorectal cancer survival. Br J Surg 2002;89:1557-62.

30. Nationaal Kompas Volksgezondheid: http://www.rivm.nl/vtv/object_document/o5666n16909.html 31. Ponz de Leon, M., G. Rossi, et al. (2007). "Epidemiology of colorectal cancer: the 21-year experience of a specialised registry." Intern Emerg Med 2(4): 269-79.

32. Sant M, Aareleid T, Berrino F et al. EUROCARE-3: survival of cancer patients diagnosed 1990-1994 – results and commentary. Annals Oncol 2003;14:v61-118.

33. Stearns, A. T., D. Hole, et al. (2007). "Comparison of breast cancer mortality rates with those of ovarian and colorectal carcinoma." Br J Surg 94(8): 957-65.

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36. Common Database: ’GDP Deflator’: http://unstats.un.org/unsd/cdb/cdb_dict_xrxx.asp?def_code=http://www.dh.gov.uk/en/Policyandguidan ce/Organisationpolicy/Financeandplanning/Allocations/DH_076547

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XI - BIOMARKERS FOR EARLY DETECTION OF COLON CANCER â&#x20AC;&#x201C; THE LAST TEN YEARS 1 - Introduction There is an increasing demand for biomarkers in colon cancer for risk assessment, early detection, prognosis, and surrogate end points. A number of biomarkers have been identified for early detection of colon cancer, although the risk factors have not been identified extensively. The major advances in understanding colorectal cancer include the identification and the involvement of APC, p53, and Ki-ras in the development and progression of the disease, the identification of the aberrant crypt foci as an early preinvasive lesion, and its relation to the development of cancer. Detecting malignant neoplasms in the early stages offers clinical advantages; therefore, the National Cancer Institute has established an Early Detection Research Network.

The emphasis of the network is on translational research and collaboration among scientists.

The identification and characterization of the genetic changes in the malignant transformation process have progressed rapidly over the last three decades (1- 4). The predominant changes include deletions, rearrangements, and mutations leading to either inactivation or activation of specific target genes (5-8). Two major classes of genes, oncogenes and suppressor genes, are involved. Oncogenes are activated or deactivated genes whose products normally promote cell growth. Products include peptide growth factors, growth factor receptors, signal transduction factors, tyrosine kinase, and transcription-regulatory proteins.

More than 30 oncogenes have been mapped within the human karyotype. Tumor suppressor genes normally regulate cell growth. These genes typically produce nuclear-regulatory proteins, which interact with other regulatory proteins to alter their activity (9,10). The frequent involvement of a limited number of oncogenes, e.g., c-myc (11-13) and ras (14-17), in a wide variety of tumor scenarios suggests, nevertheless, that the number of the most critical genes needed for the transformation process may be limited. This suggests that we might be able to develop common approaches to detect these cancers.

Recently, genes not related to the oncogenes or suppressor genes have been implicated in carcinogenesis (18,19). These genes are usually referred to as the MMR genes. Although they were found years ago in bacteria and yeast, investigators have discovered two human genes, hMSH2 and hMLH1, that are homologous with the yeast MMR genes (4,20). In addition to carcinomas of the colon, these genes have also been implicated in carcinomas occurring in other sites including the lung, urinary bladder, and ovary. These advances have fueled optimism that abnormal genes, whether acquired or inherited, may be used to assess the risk of cancer or to detect existing cancer early, even in its preclinical stages.

Knowledge of the biology of tumor progression therefore allows us to identify specific tests that are useful for early detection or screening. Molecular probes, for instance, could detect altered DNA shed into the feces (CRC), into sputum (lung cancer), and in exfoliated cells in bladder washings (bladder cancer). Correlation of the molecular alterations with demographic data, risk factors, environmental exposure, family history, and dietary history may provide important information on the etiology of cancer.

Molecular genetic alterations could also contribute toward the assessment of risk. Risk assessment is the search for risk factors that provide the earliest evidence for the risk of cancer in persons not diagnosed with the disease. Biomarkers that are predictive of risk usually trigger more aggressive interventions and surveillance. Individuals who test positive for any risk marker become candidates for an intervention or for 44

RIBOGRAMA PROJECT surveillance. The earliest risk factors are probably the inherited genetic defects, which are well demonstrated in the case of CRC (21). APC and HNPCC are associated with germ-line mutations in the APC gene and mismatch repair genes, respectively (22). Large bowel cancers often arising at a young age are found in almost 100% of individuals who inherit APC gene mutations.

2 - Biomarkers of Risk versus Early Detection Markers of risk and markers of early detection share the same outcome, namely, the incidence of disease.

However, markers of risk and markers for early detection differ in the degree of certainty they convey regarding the existence of cancer. A risk factor confers significantly less than 100% certainty of cancer within a specified time interval, whereas early detection markers confer close to 100% certainty of cancer. Risk markers indicate that cancer is more likely to occur within a specified time in persons with the marker than in the general population. Early detection markers indicate the existence of cancer or that cancer will occur with nearly a 100% certainty within a specified time interval.

From a screening perspective, all surrogate outcomes in individuals not diagnosed with cancer are risk factors.

Colonic polyps, for instance, are a surrogate end point for screening and a risk factor for colon cancer. At least three elements are necessary to use risk factors as surrogate outcomes in screening: (a) the risk factor and its detection method must be properly defined; (b) the definitive outcome of interest and a description on how to assess it should be indicated; and (c) knowledge of the strength and direction of the relationship between the surrogate outcome and the definitive outcome over a specified time interval should be known. For a risk factor to be a useful surrogate outcome, it must be strongly connected to the definitive outcome, and the probability and direction of the relationship must be known.

Several criteria must be met before biomarkers can serve as risk factors or as markers for early detection: (a) the biomarker must be differentially expressed in normal, premalignant or high-risk, and tumor tissue; (b) the marker and its assay must provide acceptable predictive accuracy for risk or for the presence of cancer; and (c) the variance of the detection tests and the intra- and interlaboratory variance must be known. For biomarkers to serve as surrogate end points in prevention interventions, it is necessary to satisfy additional criteria: (a) the marker must be a determinant of outcome; (b) the marker must be modulated by chemopreventive agents; and (c) modulation or elimination of the risk marker must correlate with a decrease in cancer incidence. Risk markers are usually used as surrogate outcomes to detect the effect of a prevention intervention more rapidly than waiting for the definitive outcome. These criteria can be tested and evaluated in animal models and in human tissue specimens. Biomarkers for colon cancer are most likely to be found in stool specimens.

3 - Biomarkers for Risk There are very few biomarkers for risk of colon cancer, such as colonic adenomas (23). Biomarkers of risk can be inherited or acquired. With the identification and characterization of inherited defective genes implicated in the etiology of CRC, it has now become possible to identify individuals predisposed to this disease through genetic testing. For this reason, widespread genetic screening will not substantially reduce the incidence of the disease unless additional genes are found. Additional genes may exist because the incidence of colon cancer is 3â&#x20AC;&#x201C;4 times higher in families with a history of this disease than in the general population (24,25). The risk of CRC begins to increase after the age of 40 years and rises sharply at the ages 50â&#x20AC;&#x201C;55 years; the risk doubles with each succeeding decade, reaching a peak by age 75 years.

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RIBOGRAMA PROJECT Models made using tissues removed from patients with hereditary CRC have indicated that hereditary CRCs follow an autosomal dominant pattern of inheritance. However, hereditary CRCs do not develop just because a single copy of a mutated gene is inherited (because the mutated gene is not sufficient by itself to cause the development of CRC). A somatic mutation must also occur in the remaining normal copy of the gene, and this mutation must occur in a cell that has the capability to proliferate. Therefore, a stem cell must have two mutated copies for the inherited gene (germ-line mutation and somatic mutation) to develop hereditary CRC. The interaction of germ-line mutations and other risk factors (such as diet) is probably due to the effects of risk factors on the induction of somatic mutations. On the other hand, FAP is a rare autosomal dominant condition (about 1 in 8000 individuals and 1% of CRC) in which patients by definition have more than 100 colorectal adenomas. FAP is associated with the development of CRC within 15 years after the adenomas begin to develop. Many of the genes and chromosomes involved in the development of CRC were identified by evaluating neoplastic and uninvolved tissues from patients with FAP (to identify chromosomal abnormalities), and the association involved genes in patients and immediate family members with this condition. Because of genetic heterogeneity and the complexity of cancer, none of these defective genes by themselves or in combination seem to be fully predictive of cancer development. Thus, new potential biomarkers need to be investigated.

The diagnosis of HNPCC requires CRC development in at least two generations and in at least three different family members. Of the three affected family members, one must be a first-degree relative of at least two other involved family members, and at least one of the tumors must have developed in a family member before the age of 50 years. A large proportion of patients with HNPCC have genetic defects in enzymes associated with DNA MMR. Inactivation of the normal allele results in genomic instability manifested by ubiquitous somatic mutations in MSI. A Western lifestyle, typical of North America, Europe, or Australia, is associated with a high risk of CRC. Kaaks et al.(26) recently conducted a case-control study of a cohort of 14,275 women in New York and observed that chronically high levels of circulating insulin and IGF-I associated with a Western lifestyle increased CRC risk, possibly by decreasing IGF-binding protein 1 and increasing the bioactivity of IGF-I. Insulin and IGF-I regulate energy metabolism, stimulate cell proliferation and anabolic processes as a function of available energy and elementary substrates (e.g., amino acids), and inhibit apoptosis. Overeating, a lack of physical activity, and obesity cause insulin resistance and hence tend to increase plasma insulin concentrations. Insulin, in turn, can increase the bioactivity of the IGF-I by inhibiting the synthesis of certain IGF-binding proteins (26).

Dietary pyrolysis product from overcooked meats may increase colorectal carcinogenesis. Dietary components that are postulated to reduce the risk of this cancer include high starch diets and high consumption of calcium and vitamins (A, B, C, D, E, β-carotene, and folate). These dietary components interact with colonic bacteria with bile acids as well as with intestinal mucin and dietary carcinogens (27).

4 - Biomarkers for Early Detection Colorectal cancer is the second most common cause of cancer death in the United States. Several studies have concluded that there is a higher incidence of CRC in persons with polyps. The term polyp is applied to any mucosal lesion that projects in the lumen of the GI tract (27). About 90% of preinvasive neoplastic lesions of the colorectum are polyps or polyp precursors (aberrant crypt foci). However, several types of polyps are not neoplastic, such as hyperplastic polyps. Therefore, neoplastic polyps are often referred to more specifically as adenomas or adenomatous polyps. Nevertheless, the term polyp is used by some clinicians or investigators to refer to neoplastic lesions.

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RIBOGRAMA PROJECT There is evidence that colon cancer progresses from normal tissue to adenoma and carcinoma through an accumulation of genetic alterations (4,28,29). These molecular genetic alterations in various types of adenomas and carcinomas offer the opportunity to detect specific tumor-related changes in DNA. In colon cancer, for example, delineation of various stages during tumor progression offers a window of opportunity to intervene in the process by detecting stage-specific molecular changes (such as change in gene expression and accumulation of mutations). To date, we know of mutations and loss of heterozygosity that affect oncogenes such as K-ras (16) and several tumor suppressor genes including p53 (10), MCC (mutated in CRC; 30), APC (31), and DCC (deleted in colon cancer; 32. Some of these genetic alterations are being tested for their potential to serve as clinical biomarkers of colon cancer development. In the following section, we discuss some potential biomarkers of colon cancer.

5 - APC Germ-line APC mutations are considered an early detection marker because nearly 100% of individuals with the mutation will develop colon cancer in the future. For example, RFLP of chromosome 5q21â&#x20AC;&#x201C;22 (for loss of the APC gene) has been shown to be useful for premorbid diagnosis and counseling in FAP. Although APC is a rare disorder, which occurs in only 1 in 500 persons and accounts for less than 1% of all cases of colonic carcinoma, mutations in the APC gene occur in 60% of patients with colorectal carcinoma and are thought to be the earliest genetic abnormalities in the progression of colorectal carcinoma. The I1307K mutation was found in approximately 6% of the Ashkenazi Jewish population and is associated with elevated risk of CRC (22). PCR-based tests are used to detect these mutations (33).

6 - MMR Molecular research has identified a new family of genes, commonly known as MMR genes, that predisposes individuals to colon cancer. A number of genes are involved in MMR, such as hMSH2, hMLH1, hPMS2, hMSH3, and hMSH6 (for references, see 27). For instance, HNPCC, popularly known as Lynch Syndrome (34,35), is found in as many as 1 in 200 individuals in the Western world and has been the subject of intense study for the involvement of MMR genes. Mutations of these genes produce instability of microsatellite sequences. Loss of MMR leads to greatly elevated (100â&#x20AC;&#x201C;1000-fold) frequencies of point mutations (mutator phenotype) and MSI frameshifts. MSI occurs in most CRCs from patients with HNPCC and therefore has been used as a biomarker for the detection of the HNPCC syndrome.

7 - K-ras More than 50% of patients with adenocarcinoma of the colon carry a mutant allele of K-ras genes. The high frequency of this mutation and its early appearance in colon cancer point to its potential to serve as a biomarker for early detection (3,36,37). Because an apparently satisfactory assay for ras mutations in feces has already been developed (3,37), the focus is on sensitive detection of this molecular marker in colonic effluent samples that are routinely obtained before colonoscopy. More importantly, stool samples contain sufficient amounts of DNA for PCR amplification and represent the colonic cell spectrum (36,38,39). Among various methods that have been developed for facilitating the screening of point mutations in human genomic DNA, PCR-PIRA is of particular interest due to its practicality and short procedure allowing detection of point mutations by simple restriction enzyme digestion directly after PCR amplification. However, one limitation of the PCR-PIRA method is the absence of restriction sites in the region of detection; thus, creation of the recognition site in primers has been introduced. However, false positive results generated from undigested normal DNA sequence are always obtained. This effect is compounded when it is used to analyze mixed cell 47

RIBOGRAMA PROJECT populations in paraffin-embedded sections. Assay of a mutant band generated from normal DNA by densitometric quantitation enabled the determination of background values and thereby eliminated false positive results. Samples with higher ratios between mutant and normal bands than the background one after the first PCR-PIRA would be subjected to the second PCR-PIRA to confirm the results.

8 - p53 For p53, unless the mutation is inherited, the situation is somewhat different from that of ras, in that an acquired defect of p53 or loss of the wild-type allele generally tends to be a later event in colorectal carcinogenesis, probably occurring near the time of invasion (32). Data from the literature suggest that at least a subset of familial cases of GI malignancies, sarcomas, and breast carcinoma is associated with inherited p53 mutations (40-42) and that a family history with these cancers in the pedigree may identify a group of patients in whom this molecular risk factor could be detected and those patients logically pursued with periodic screening. Genomic DNA of 118 colon cancer patients was analyzed for mutation detection in exons 4â&#x20AC;&#x201C;8 of the p53 gene and codons 12/13 and 61 of the K-ras gene by single-strand conformational polymorphism and direct sequencing (36). The production of p53 and K-ras proteins was studied by immunohistochemistry. The overall frequency of mutations in the p53 gene was 35%, but the true frequency appeared to be higher (up to 56%). In the K-ras gene, the mutation frequency (15%) was significantly lower than that reported for colon cancer. In the p53 gene, the mutation frequency increased significantly with patient age. In a high proportion of patients (14%), the rectal tumors contained small subclones of tumor cells that displayed extremely rare mutations at codons 110 and 232 of the p53 gene. Hot spot codon 175 mutations were significantly less common in rectal cancer than in cancer of the colon. p53 mutations were tested in DNA samples using PCRbased approaches.

9 - Kinases The family of protein tyrosine kinases includes many known oncogenes and growth factor receptors that have been implicated in the pathogenesis of human cancers (43-46). The tyrosine kinases exert their cellular growth control by phosphorylating tyrosine residues on proteins. The prototype tyrosine kinase, the product of pp60csrc oncogene, is an example of an intracellular protein kinase. This kinase has been shown to be activated at an early stage of colon tumorigenesis (47). Other protein tyrosine kinases are those that function as cell surface receptors, such as epidermal growth factor receptor and HER-2/neu. HER-2/neu has been shown to be associated with the earliest events in human breast carcinoma, as well as with the later stages of this disease (47). The focus of the study was on early detection by the following algorithm: tyrosine kinases expressed by (a) normal colonic mucosa, (b) colonic polyps (both tubular and villous adenomas), (c) primary colon cancers, and (d) metastatic colon cancer were identified and catalogued. A kinase that is expressed in both normal and neoplastic colonic epithelium would not be expected to have a role in tumorigenesis and would not be studied further. A kinase with higher expression levels in colon polyps or primary colon cancers than in normal colonic mucosa would be a potential biomarker for the development of colon cancer. Similarly, a marker expressed by villous adenomas, as opposed to tubular adenomas, would be another candidate for a marker of malignant progression, given the different malignant potential of these two precancerous lesions.

Immunohistochemical approaches were applied to detect these markers in tissues, and immunoassays were used in serum samples.

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RIBOGRAMA PROJECT 10 - Serum Markers Several markers, including serum markers, have been used to detect early onset and the recurrence of colorectal tumors: (a) early detection of colorectal neoplasia by detection in feces of mutations in ras or p53 or of changes in microsatellites of DNA (37,48); (b) targeting of carcinoembryonic antigens and TAG-72 for immunotherapy or for tumor vaccines; (c) use of markers for atypical mucins (e.g., TAG-72) and neuroendocrine markers (neuron-specific enolase or argyrophilic stains) in diagnosis to separate colorectal carcinoma from carcinoid tumors; and (d) use of changes in proliferation and cytomorphometric parameters in pre- and postchemoprevention colorectal biopsies as SEBs. In practical use of biomarkers to identify colorectal tumors (which may have more aggressive clinical behavior), the first step would be to characterize the general expression of biomarkers throughout the development of colorectal tumors to correlate these differences independent of tumor stage. Monoclonal antibody B72.3 binds to TAG-72 antigen present in the serum of CRC patients. Petersen et al (49) detected TAG-72 in 86% of serum samples using an enzyme immunoassay.

Understanding the limitations in the detection of antigen expression in archival tissues is necessary to use biomarkers (for example, the sensitivity and specificities of antibodies to antigens in paraffin-embedded sections and the effects of tissue fixation and tissue processing on the detection of specific antigens). Failure to recognize these issues may result in false negative results, leading to inappropriate exclusion of patients from treatment or inappropriate diagnosis of protocols and to confounding comparisons of populations defined by these markers.

11 - L-DNA The high-integrity DNA or L-DNA has proven to be the most informative component marker of the stool DNA assay panel, and the marker has been found in 61% of colon cancers(3). Longer template DNA is an epigenetic phenomenon consistent with the known abrogation of apoptosis. DNA in normal stools would exist primarily in fragments (180–200 bp), whereas that of colon cancer patient should contain a subset of L-DNA arising from dysplastic cells. For the assay, the primers were designed to give products much longer than 200 bp, and thus only longer DNA was used as the template. Fragments as large as 2.4 kb have been observed by following this assay.

12 - β-Catenin An increase in cytoplasmic β-catenin levels and subsequent β-catenin/Tcf-lymphoid enhancer factor complex formation are believed to be important events in the early stage of colonic carcinogenesis (4,50-52). This complex binds to the DNA and results in overexpression of prostaglandin endoperoxide H synthase-2 (COX2), induced by nitric oxide. Recently, Miyaki et al. (53) reported frequent mutations of the β-catenin and APC genes in primary colorectal tumors from patients with HNPCC. Their previous study detected a low frequency of APC gene mutation (21%) in colorectal tumors from HNPCC patients, in contrast to a high frequency of APC gene alteration (>70%) in non-HNPCC tumors. Because both β-catenin and APC gene mutations have recently been shown to activate the same signaling pathway, Miyaki’s group analyzed β-catenin mutation in HNPCC tumors. A notable frequency of β-catenin gene mutation (43%, 12 of 28) was found to occur in HNPCC colorectal tumors. However, β-catenin mutations were not detected in tumors with APC mutations. All β-catenin mutations detected in HNPCC tumors existed within the regulatory domain of β-catenin.

Immunohistochemical staining of tumors with this mutation showed accumulation of β-catenin protein in nuclei.

These and previous data from their laboratory suggest that activation of the β-catenin-Tcf signaling pathway, through either β-catenin or APC mutation, contributes to HNPCC carcinogenesis in approximately 65% of cases. β-Catenin is assayed in serum by immunological methods.

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RIBOGRAMA PROJECT 13 - bcl-2 Gene The product of the bcl-2 gene (Bcl-2) is a 26-kDa protein that inhibits apoptosis. Several studies have evaluated expression of Bcl-2 protein using immunohistochemistry (54-59). Expression of Bcl-2 in CRCs has been demonstrated as a favorable prognostic factor in Austrian (60) and German populations (58). However, such prognostic significance could not be seen in a large United States population with colorectal carcinomas, although the incidence of Bcl-2-expressing tumors was similar to that seen in the European studies. Results from other laboratories (61) indicated that patients with colorectal carcinomas that express Bcl-2 have a better clinical outcome than patients with CRCs that do not express Bcl-2 (62). The reasons for the reported differences in the prognostic importance of Bcl-2 are not known, but they may be due to geographic and/or ethnic/racial variations in the patient populations of the various studies.

14 - Proliferation in Aberrant Crypt Foci Proliferative cells are typically restricted to the lower two-thirds of the normal colorectal crypt. An increase in cellular proliferation has been observed in adenomas as compared with benign epithelium, with the highest proliferation seen in adenomas with high-grade dysplasia. In addition, a shift of proliferation toward the luminal surface of the epithelium occurs in adenomas as well as aberrant crypt foci. Increased proliferation as well as an upward shift of the proliferative component has also been observed in the â&#x20AC;&#x153;transitionalâ&#x20AC;? epithelium adjacent to adenomas or colorectal carcinomas. Furthermore, a shift or expansion of the proliferating zone toward the luminal surface has been reported in uninvolved colonic epithelium of patients with adenomatous polyps or colorectal carcinomas; however, a large study failed to demonstrate a difference in proliferation as measured

by Ki-67 in the benign colorectal epithelium in patients with colorectal carcinomas as compared with controls (63). Although controversial, the increased proliferation observed in mucosa distant from CRC has been interpreted as evidence that the entire colorectum may undergo specific premalignant changes, such as the field effect. Such changes in proliferation could be the result of stimulation by paracrine factors released by malignant cells or colonization of a large proportion of the colorectal epithelium by a clonal expansion. An increase in proliferation in the normal colorectal crypts of individuals with a high risk of CRC based on family history was demonstrated by Rooney et al (64). Other investigators described a shift in proliferation from the base toward the surface in the intervening benign mucosa (in patients with FAP) (65) . Another study demonstrated a shift in the proliferative cells toward the lumen in uninvolved colonic epithelium of FAP patients(66). Similarly, increased proliferative rates and an expansion of the proliferative zone to the luminal surface of the epithelium have been observed in the colonic epithelium of HNPCC patients (34,67,68).

15 - Cyclin D1 Several studies have demonstrated increased expression of the cyclin D1 protein in 30â&#x20AC;&#x201C;45% of CRCs when compared with uninvolved epithelium (69,70). In addition, increased expression of cyclin D1 protein was observed in 34% of adenomatous polyps (69). Cyclin D1 is measured in serum by immunological assays.

16 - Cyclin E1 Increased cyclin E1 levels were observed in colorectal carcinomas as compared with uninvolved colonic mucosa (in 90% of patients; 71). A direct association of increased cyclin E1 levels with the graded atypia within adenomas and with local invasiveness as well as proliferation potential of CRC (72,73). Amplification of cyclin E1 has been reported in 9% of primary CRCs (73).

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RIBOGRAMA PROJECT 17 - SMAD4 Woodford-Richens et al (23) demonstrated allelic loss at SMAD4 in polyps from JPS patients and used fluorescent in situ hybridization to demonstrate clonal origin of the epithelium. Germ-line mutations in SMAD4 (DPC4) account for about one-third of this population. They found allele loss at the SMAD4 locus on 18q in polyps from JPS individuals with a germ-line SMAD4 mutation, showing that SMAD4 acts as a tumor suppressor gene in JPS polyps. These mutations were detected in DNA (isolated from cells) using PCR-based methods (4).

18 - Panel of Markers Ahlquist’s group (3,74) has found that DNA released from cells that regularly slough into the stool can be used to identify cancer or precancerous polyps in the colon. They have developed a multitarget DNA assay panel that shows a remarkably higher sensitivity and specificity than traditional fecal blood tests (3). The multitarget DNA assay panel targets point mutations at any of 15 mutational hot spots on K-ras, p53, and APC genes; Bat-26, a microsatellite instability marker; and L-DNA. In the first pilot study on stools from 22 patients with CRC, 11 patients with adenomas (≥1 cm) and 28 colonoscopically normal controls revealed a sensitivity for cancer of 91% and a sensitivity for adenomas of 73% with a specificity of 100%. Thus, an assay of altered DNA holds promise as a stool screening approach for colorectal neoplasia. The first group (85% of tumors) exhibits chromosomal instability but stability of microsatellite DNA, and the second group (15% of tumors) exhibits MSI. The components of the second group consist of DNA MMR genes, TGF-β-RII, BAX, IGF-IIR, E2F4, and Tcf-4 (4).

19 - Biomarkers as Intermediate End Points in Colorectal Cancer Prevention Although hyperproliferation has been postulated as a relatively early event in the pathway to colorectal carcinoma, it may not be a necessary step, and incorrect inferences may be drawn from a study that uses a proliferation marker as the sole surrogate end point for invasive cancer. An example of this is an adenoma recurrence trial at Dartmouth Medical School. Calcium had no effect on proliferation markers but showed a modest reduction in adenoma recurrence in the calcium arm (29). Alternatively, the effect of an intervention agent on hyperproliferation may be counteracted by its effect on another pathway, so there is no net effect on colon cancer. Surrogate end points must be a determinate of outcome.

The 36-kDa PCNA is an example of a colorectal cell proliferation marker. PCNA can be detected in cells in rectal mucosal crypts by immunohistochemical analysis. The PCNA-labeled cells are indicative of dividing/proliferating cells and mainly reflect DNA synthesis. Various indices of the total proportion of crypt cells that are labeled as well as indices reflecting the location of proliferating cells within the crypt can be determined.

Even when a marker meets these criteria when tested with one agent, it cannot be used with confidence in the evaluation of another agent because the active agent may affect both a pathway through the surrogate and a pathway through another marker. Even if the effect of the active agent on the alternative pathway does not offset its effect on the surrogate, the same cannot be said to be necessarily true for another agent. One could be more confident if the two agents under consideration operated through exactly the same pathway, but new agents, even those structurally similar to proven agents, have unanticipated actions and therefore have unintended consequences.

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RIBOGRAMA PROJECT 20 - Adenomas as a Logical Surrogate End Point Adenomas satisfy the criteria for a surrogate end point for prevention studies and for early detection. They are a risk factor for colon cancer and a surrogate outcome for screening because most tumors appear to arise from polyps. Studies indicate that they may be modulated by chemopreventive agents, although it remains to be determined whether chemical modulation leads to a reduction in the incidence of colon cancer. Adenomas can be identified and removed through colonoscopy or sigmoidoscopy. Thus, as a risk factor, the polyp serves to initiate a preventive intervention. Although relatively common, the majority of adenomas do not progress to invasive cancer.

21 - Nonsteroidal Chemopreventive Agents and Biomarkers SEBs have become widely used in short-term cancer chemoprevention trials in place of cancer end points.

Here we have discussed the criteria relevant to the selection and validation of SEBs for colon cancer risk and the use of SEBs in colon cancer chemoprevention trials. As with a number of other cancers, colon carcinogenesis is the result of a multistep process in which an increasing number of alterations, including specific gene mutations, occur as cells progress from a normal state to a precancerous state of increasing size and dysplasia to cancer and finally to metastatic disease. Ideally, a SEB would show differential expression between the various phases of colon carcinogenesis (i.e., normal, premalignant, and malignant tissues) and would be associated with risk of colon cancer. Some SEBs that do not meet these criteria may still be useful for demonstrating the effect of a particular agent. It is also necessary that a SEB be measured in tissues (or other sample material) accessible for multiple and sequential sampling and allow for the development of appropriate quality control procedures. Some SEBs must have the potential for modulation by chemopreventive agents. Validation of SEBs for use in chemoprevention studies requires that a relationship between the marker and subsequent risk of cancer be established. Also, the assay reliability and accuracy for each SEB must be determined adequately in well-designed prospective studies.

The epidemiological studies that have detected a 40â&#x20AC;&#x201C;50% decrease in risk of CRC in individuals who regularly use aspirin and other NSAIDs such as celecoxib could not have included because of such regular NSAID use (29,75-78). NSAIDs appear to act via induction of apoptosis, programmed cell death, as potential CRC chemopreventive agents. NSAIDs can alter the production of different metabolites of polyunsaturated fatty acids (linoleic and arachidonic acids) through effects on lipoxygenases and COXs. 15-Lipoxygenase-1 is the main enzyme for metabolizing colonic linoleic acid to 13-S-hydroxyoctadecadienoic acid, which induces apoptosis. Clinical trials with NSAIDs in patients with FAP have demonstrated that treatment with NSAIDs caused regression of preexisting adenomas. The surrogate end point in these studies was polyp burden (77).

Steinbach et al. (75) investigated the effect of celecoxib, a selective inhibitor of COX-2, on colorectal polyps in (77) 77 patients with FAP. Patients underwent endoscopy at the beginning and end of the study. Six months of treatment resulted in a significant reduction in the number of colorectal polyps.

Several, but not all, epidemiological studies have reported a reduction in colon cancer incidence associated with the use of aspirin (76-78). Several cohort studies suggested a preventive effect of aspirin. Among a group of over 600,000 adults enrolled in an American Cancer Society study, mortality in regular users of aspirin was about 40% lower for cancers of the colon and rectum (78,79). In a study of over 11,000 men and women in Sweden with rheumatoid arthritis (and presumably ingesting NSAIDs), colon cancer incidence was 37% lower than predicted from cancer registry data, and rectal cancer was 28% lower than predicted from cancer registry data (80). In a report from the Health Professionals Follow-up Study of 47,000 males, regular use of aspirin (at least 2 times/week) was associated with a 30% overall reduction in CRC including a 50% reduction in advanced cases (81). In contrast to the results from cohort studies, the Physiciansâ&#x20AC;&#x2122; Health Study, which was a 52

RIBOGRAMA PROJECT randomized controlled trial designed to test whether low-dose aspirin (325 mg of aspirin every other day) would reduce cardiovascular disease mortality, did not show a reduction in invasive cancers or adenomas after 4.5 years among the participants assigned to receive aspirin (82). The interpretation of the data is complicated by the possibility that prevalent tumors at the time of randomization could have been detected early and by the differences in age among the study populations (83,84). Several studies conducted in a rigorous manner have demonstrated the effectiveness of sulindac in reducing the size and number of adenomas in familial polyposis (85,86).

The NSAID piroxicam, at a dose of 20 mg/day, reduced mean rectal prostaglandin concentration by 50% in individuals with a history of adenomas (87). In this study, mucosal prostaglandin concentration was measured as the end point. Preclinical efficacy studies have provided scientifically sound evidence as to how NSAIDs act to retard, block, or reverse colonic carcinogenesis. Equally exciting are opportunities for effective chemoprevention with selective COX-2 inhibitors in a variety of animal models of colon cancer. Selective COX2 inhibitors such as celecoxib have been proven to be effective chemopreventive agents against colonic carcinogenesis with minimal GI toxicity. Similarly, the short-chain fatty acid butyrate, produced by microbial fermentation of dietary fiber in the large intestine, has been used to intervene in major pathways of colonic epithelial cell maturation, such as cell cycle arrest, lineage-specific differentiation, and apoptosis. β-Catenin and polyp burden were used as end point markers in these studies (76). The potential for the use of NSAIDs as a primary prevention measure is intriguing. However, there are several unresolved issues that mitigate against making general recommendations for their use. These include a paucity of knowledge about the proper dose and duration for these agents. There is also concern about whether the potential preventive benefits would balance such long-term risks as GI ulceration and hemorrhagic shock for the average-risk individual (88).

It will be necessary to construct a link network of centers of expertise in tumor biology, diagnostic technologies, and clinical trial methodology in academia and industry to develop high-throughput assays suitable for clinical application. These laboratories/centers will cover a range of study designs, technology development, and innovative approaches from genomics to proteomics in pursuit of developing molecular, genetic, and biological markers for earlier cancer detection and for the identification of high-risk subjects.

Footnotes Abbreviations used: MMR, mismatch repair; APC, adenomatous polyposis coli; HNPCC, hereditary nonpolyposis colorectal cancer; FAP, familial adenomatous polyposis; IGF, insulin-like growth factor; MSI, microsatellite instability; PCR-PIRA, PCR-primer-introduced restriction analysis; GI, gastrointestinal; L-DNA, long DNA; Tcf, T-cell factor; COX, cyclooxygenase; JPS, juvenile polyposis; PCNA, proliferating cell nuclear antigen; SEB, surrogate end point biomarker; NSAID, nonsteroidal anti-inflammatory drug; CRC, colorectal cancer.

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54

RIBOGRAMA PROJECT 22. Syngal S., Schrag D., Falchuk M., Tung N., Farraye F. A., Chung D., Wright M., Whetsell A., Miller G., Garber J. E. Phenotypic characteristics associated with the APC gene I1307K mutation in Ashkenazi Jewish patients with colorectal polyps. JAMA, 16: 857-860, 2000.

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35. Lynch H. T., Lemon S. J., Karr B., Franklin B., Lynch J. F., Watson P., Tinley S., Lerman C., Carter C. Etiology, natural history, management and molecular genetics of hereditary nonpolyposis colorectal cancer (Lynch syndromes): genetic counseling implications. Cancer Epidemiol. Biomark. Prev., 6: 987-991, 1997.

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RIBOGRAMA PROJECT 40. Malkin D., Li F. P., Strong L. C., Fraumeni J. F., Jr., Nelson C. E., Kim D. H., Kassel J., Gryka M. A., Bischoff F. Z., Tainsky M. A. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science (Wash. DC), 250: 1233-1238, 1990.

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42. Law J. C., Strong L. C., Chidambaran A., Ferrell R. E. A germ line mutation in exon 5 of the p53 gene in an extended cancer family. Cancer Res., 51: 6385-6387, 1991.

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Br. J. Cancer, 83: 668-673, 2000.

44. Slamon D. J., Clark G. M., Wong S. G., Levin W. J., Ullrich A., McGuire W. L. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science (Wash.

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45. Sainsbury J. R. C., Farndon J. R., Needham G. K., Malcolm A. J., Harris A. L. Epidermal-growth-factor receptor status as predictor of early recurrence of and death from breast cancer. Lancet, 1: 13981402, 1987.

46. Mishra L., Bass B., Ooi B. S., Sidawy A., Korman L. Role of insulin-like growth factor-I (IGF-I) receptor, IGF-I, and IGF binding protein-2 in human colorectal cancers. Growth. Horm. IGF Res., 8: 473-479, 1998.

47. Cartwright C. A., Kamps M. P., Meisler A. I., Pipas J. M., Eckhart W. pp60c-src activation in human colon carcinoma. J. Clin. Investig., 83: 2025-2033, 1989.

48. Hasegawa Y., Takeda S., Ichii S., Koizumi K., Maruyama M., Fujii A., Ohta H., Nakajima T., Okuda M., Baba S. Detection of K-ras mutations in DNAs isolated from feces of patients with colorectal tumors by mutant-allele-specific amplification (MASA). Oncogene, 10: 1441-1445, 1995.

49. Petersen B. M., Jr., Bass B. L., Bates H. R., Chandeysson P. L., Harmon J. W. Use of the radiolabeled murine monoclonal antibody, 111In-CYT-103, in the management of colon cancer. Am. J. Surg., 165: 137-142, 1993.

50. Korineck V., Barker N., Morin P. J., Van Wichen D., De Weger R., Kinzler K. W., Vogelstein B., Clevers H. Constitutive transcriptional activation by β catenin-Tef complex in APC−/− colon carcinoma.

Science (Wash. DC), 275: 1784-1787, 1997.

51. Morin P. J., Sparks A. B., Krinek V., Barker N., Clevers H., Vogelstein B., Kinzler K. W. Activation of βcatenin-Tcf signaling in colon cancer by mutations in β-catenin or APC. Science (Wash. DC), 275: 1787-1790, 1997.

52. Tetsu O., McCormick F. β-Catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature (Lond.), 398: 422-426, 1999.

53. Miyaki M., Iijima T., Kimura J., Yasuno M., Mori T., Hayashi Y., Koike M., Shitara N., Iwama T., Kuroki T. Frequent mutation of β-catenin and APC genes in primary colorectal tumors from patients with hereditary nonpolyposis colorectal cancer. Cancer Res., 59: 4506-4509, 1999.

54. Hague A., Moorghen M., Hicks D., Chapman M., Paraskeva C. Bcl-2 expression in human colorectal adenomas and carcinomas. Oncogene, 9: 3367-3370, 1994.

55. Bosari S., Moneghimi L., Graziani D., Lee A. K., Murray J. J., Coggi G., Viale G. M. Bcl-2 oncoprotein in colorectal hyperplastic polyps, adenomas, and adenocarcinomas. Hum. Pathol. 26: 534-540, 1995.

56. Sinicrope F. A., Ruan S. B., Cleary K. R., Stephens L. C., Lee J. J., Levin B. Bcl-2 and p53 oncoprotein expression during colorectal tumorigenesis. Cancer Res., 55: 237-241, 1995.

57. Watson A. J. M., Merritt A. J., Jones L. S., Askew J. N., Anderson E., Becciolini A., Balzi M., Potten C. S., Hickman J. A. Evidence of reciprocity of bcl-2 and p53 expression in human colorectal adenocarcinomas and carcinomas. Br. J. Cancer, 73: 889-895, 1996.

58. Baretton G. B., Diebold G., Christoforis G., Vogt M., Muller C., Dopfer K., Schneiderbanger K., Schmidt M., Lohrs U. Apoptosis and immunohistochemical bcl-2 expression in colorectal adenomas and carcinomas. Cancer (Phila.), 77: 255-264, 1996.

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RIBOGRAMA PROJECT 59. Kaklamanis L., Savage A., Mortensen N., Tsiotos P., Doussi-Anagnostopoulou I., Biddolph S., Whitehouse R., Harris A. L., Gatter K. C. Early expression of bcl-2 protein in the adenoma-carcinoma sequence of colorectal neoplasia. J. Pathol., 179: 10-14, 1996.

60. Ofner D., Riehemann K., Maier H., Reidmann B., Nehoda H., Totsch M., Bocker W., Jasani B., Schmid K. W. Immunohistochemically detectable Bcl-2 expression in colorectal carcinoma: correlation with tumor stage and patient survival. Br. J. Cancer, 72: 981-985, 1995.

61. Sinicrope F. A., Hart J., Michelassi F., Lee J. J. Prognostic value of bcl-2 oncoprotein expression in stage II colon carcinoma. Clin. Cancer Res., 1: 1103-1110, 1995.

62. Manne U., Myers R. B., Moron C., Poczatek R. B., Dillard S., Weiss H., Brown D., Srivastava S., Grizzle W. E. Prognostic significance of Bcl-2 expression and p53 nuclear accumulation in colorectal adenocarcinoma. Int. J. Cancer, 74: 346-358, 1997.

63. Wong A. J., Kohn G. J., Scwartz B. H., Ruebner B. H., Lawson M. J. Colorectal cancer and noncancer patients have similar labeling indices by microscopy and computed image analysis. Hum. Pathol. 26: 1329-1332, 1995.

64. Rooney P. S., Robinson M. H. E., Clarke P. A., Hardcastle J. D., Armitage N. C. Individuals with a strong family history of colorectal cancer demonstrate abnormal rectal mucosal proliferation. Br. J. Surg., 80: 249-251, 1993.

65. Deschner E. E., Lipkin M. Proliferative patterns in colonic mucosa in familial polyposis. Cancer (Phila.), 35: 413-418, 1975.

66. Potter C. S., Kellett M., Rew D. A., Roberts S. A. Proliferation in human gastrointestinal epithelium using bromodeoxyuridine in vivo: data for different sites, proximity to a tumor, and polyposis coli. Gut, 33: 534-539, 1992.

67. Lynch H. T., Schuelke G. S., Kimberling W. J., Albano W. A., Lynch J. F., Biscone K. A., Lipkin M. L., Deschner E. E., Mikol Y. B., Sandberg A. A., et al Hereditary nonpolyposis colorectal cancer (Lynch syndromes I and II). II. Biomarker studies. Dis. Colon Rectum, 31: 372-377, 1988.

68. Jass J. R., Ajioka Y., Radojkovic M., Allison L. J., Lane M. R. Failure to detect colonic mucosal hyperproliferation in mutation positive members of a family with hereditary non-polyposis colorectal cancer. Histopathology, 30: 201-207, 1997.

69. Arber N., Hibshoosh H., Moss S. F., Sutter T., Zhang Y., Begg M., Wang S., Weinstein I. B., Holt P. R. Increased expression of cyclin D1 is an early event in multistage colorectal carcinogenesis.

Gastroenterology, 110: 669-674, 1996.

70. Bartkova J., Lukas J., Strauss M., Bartek J. The prad-1/cyclin D1 oncogene product accumulates aberrantly in a subset of colorectal carcinomas. Int. J. Cancer, 58: 568-573, 1994.

71. Wang A., Yoshimi N., Suzui M., Yamaouchi A., Tarao M., Mori H. Different expression patterns of cyclins A, D1 and E in human colorectal cancer. J. Cancer Res. Clin. Oncol. 122: 122-126, 1996.

72. Yasui W., Kuniyasu H., Yokozaki H., Semba S., Shimamoto F., Tahara E. Expression of cyclin E in colorectal adenomas and adenocarcinomas: correlation with expression of Ki67 antigen and p53 protein. Virchows Arch., 429: 13-19, 1996.

73. Kitahara K., Yasui W., Kunyasu H., Yokozaki H., Akama Y., Yunotani S., Hisatsugu T., Tahara E. Concurrent amplification of cyclin E and cdk2 genes in colorectal carcinomas. Int. J. Cancer, 62: 2528, 1995.

74. Hara A. K., Johnson C. D., Reed J. E., Ahlquist D. A., Nelson H., Ehman R. L., McCollough C. H., Ilstrup D. M. Detection of colorectal polyps by computed tomographic colography: feasibility of a novel technique. Gastroenterology, 110: 284-290, 1996.

75. Steinbach G., Lynch P. M., Phillips R. K., Wallace M. H., Hawk E., Gordon G. B., Wakabayashi N., Saunders B., Shen Y., Fujimura T., Su L. K., Levin B. The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N. Engl. J. Med., 342: 1946-1952, 2000.

76. Mariadason J. M., Corner G. A., Augenlicht L. H. Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: comparison with trichostatin A, sulindac, and curcumin and implications for chemoprevention of colon cancer. Cancer Res., 60: 4561-4572, 2000.

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RIBOGRAMA PROJECT 77. Reddy B. S., Rao C. V. Colon cancer: a role for cyclo-oxygenase-2-specific nonsteroidal antiinflammatory drugs. Drugs Aging, 16: 329-334, 2000.

78. Thun M. J., Namboodiri M. M., Health C. W. Aspirin use and reduced risk of fatal colon cancer. N. Engl. J. Med., 325: 1593-1596, 1991.

79. Thun M. J., Namboodiri M. M., Calle E. E., Flanders W. D., Heath C. W., Jr. Aspirin use and risk of fatal cancer. Cancer Res., 53: 1322-1327, 1993.

80. Gridley G., McLaughlin J. K., Ekbom A., Klareskog L., Adami H. O., Hacker D. G., Hoover R., Fraumeni J. F., Jr. Incidence of cancer among patients with rheumatoid arthritis. J. Natl. Cancer Inst.

(Bethesda), 85: 307-311, 1993.

81. Giovannucci E., Rimm E. B., Stampfer M. J., Colditz G. A., Ascherio A., Willett W. C. Aspirin use and the risk for colorectal cancer and adenoma in male health professionals. Ann. Intern. Med., 121: 241246, 1994.

82. Gann P. H., Manson J. E., Glynn R. J., Buring J. E., Hennekens C. H. Low-dose aspirin and incidence of colorectal tumors in a randomized trial. J. Natl. Cancer Inst. (Bethesda), 85: 1220-1224, 1993.

83. Greenberg E. R., Baron J. A. Prospects for preventing colorectal cancer death. J. Natl. Cancer Inst.

(Bethesda), 85: 1182-1184, 1993.

84. Paganini-Hill A. Aspirin and the prevention of colorectal cancer: a review of the evidence. Semin.

Surg. Oncol., 10: 158-164, 1994.

85. Labayle D., Fischer D., Vielh P., Drouhin F., Pariente A., Bories C., Duhamel O., Trousset M., and Attali P. Sulindac causes regression of rectal polyps in familial adenomatous polyposis.

Gastroenterology, 101: 635-639, 1991.

86. Giardiello F. M., Hamilton S. R., Krush A. J., Piantadosi S., Hylind L. M., Celano P., Booker S. V., Robinson C. R., Offerhaus G. J. Treatment of colonic and rectal adenomas with sulindac in familial adenomatous polyposis. N. Engl. J. Med., 328: 1313-1316, 1993.

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88. Sandler R. S. Aspirin and other nonsteroidal anti-inflammatory agents in the prevention of colorectal cancer. Cancer: Principles and Practice of Oncology Updates. 11: 1-14, 1997.

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XII - POTENTIAL OF THE RIBOGRAMA PROJECT We intend to present a research and health project on cancer (RIBOGRAMA PROJECT), for an innovative method of early diagnosis of colorectal cancer (and cancers of other organs and systems), which was developed within the studies of a PhD thesis at the University Complutense of Madrid. The scope and depth of this new method of research on cancer RIBOGRAMA is exposed in a brief monograph on the concept and applications, which is part of this presentation and is an instrument of dissemination and promotional marketing of potentialities of this method for monitoring the changes of the phenotype of eukaryotic cells under the stimuli of the environment where they are. For example, to speak of the great technical and economic potential of this new method of investigation and monitoring of cancer of colon and rectum, we take the sample of the whole population of people living in Europe, corresponding to European Union countries, representing almost a 500 million people.

The sporadic colorectal cancer is a disease, which begins substantially at about the 50 years of age. In statistical terms, the population of Europe with over 50 years of age has an expression roughly 50% of its total.

Thus, broadly speaking, about half the European population will be older than 50 years, which therefore correspond to a target/universe to study (the colorectal cancer) corresponding to 250 million. If in these people there is an adherence to the RIBOGRAMA test execution at about 10% of total value, given its great simplicity and strong powers of persuasion and information about the disease of colorectal cancer, then we have a scenario testing around 25 million people, which is carried out every 6 months, will involve the execution of 50 million tests per year!!.

According to Eurostat, annually die in the European Community, more than 200 000 people of colorectal cancer. Given these figures and given the great simplicity of implementation and interpretation of test results RIBOGRAMA, implying an early preventative health politic of authorities of each country from the perspective of public health measures on the part of the governments, it will not be difficult to imagine how easy it will reduce the actual number of deaths at least to half of those referred 200,000 deaths. This is the design of this RIBOGRAMA PROJECT. Beyond the European continent, we add the same reasoning for calculating other continents and large countries, it is easy to conclude that we are facing an exponential growth in business, and that without putting the scenario if the RIBOGRAMA method is also used in the study of cancer regarding other organs and body systems, which would add to other areas in Life Sciences and Biosciences.

The beginning and development of a project with such dimensions is only possible with a project with financial support and scientific coordination by the author and owner of the intellectual authorship of the proposal putted in his PhD thesis (at Universidad Complutense de Madrid) whose idea, already patented, will be of great utility, and may have an economic and financial returns within set between 3 and 5 years, with a clear advantage for the States, to being able to achieve in a few more years, an amount that will exceed two thousand five hundred million Euros/year, given the savings in loss of life and cost savings for the Ministry of Health and the Ministry of Labour and Social Solidarity, given the savings in costs and gains in profits derived, directly or indirectly resulting from cancers of other organs and systems, which would also be the target of the method RIBOGRAMA, both in early diagnosis, either in the direction of anti-cancer treatment.

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RIBOGRAMA PROJECT

Briefly, it can be said that we have a method eminently practical and of great economic and social impact. For the patient and the doctor, the method allows to see the cancer of colon and rectum as a chronic disease that can be controlled, as in diseases such as diabetes, dyslipidemia, in a large scale without using invasive blindly methodologies, unless the patient reaches the doctor with manifest symptoms of colorectal cancer.

In this methodology there are practical and objective forces which allow placing patients with high curves of RIBOGRAMA in risk groups, which then have a formal indication to be studied and treated by endoscopic.

After all, there are "papers" published that speak in sharp decline, or disappearance of polyps with medical treatment on the basis of a diet and prescription of medication already licensed for cardiovascular protection and anti-inflammatory activity. With this method it is possible to significantly lower the number of deaths from colorectal cancer (and other cancers) in the countries, if we adopt sanitary measures for screening and diagnosis with the RIBOGRAMA METHOD, because the extent of populations to follow the RIBOGRAMA METHOD will be quite high.

In the case of colorectal cancer the extent of adhesion to the method will be good given the following sum of twelve "friendly" factors: 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

No invasive act; There is no necessity for the patient to handle its own feces during the harvest; No fecal odor annoyance since the collector of feces is designed industrially accordingly; No bowel preparation is necessary; Enemas are not needed cleaning; It does not require the use of cathartics; It is not necessary to shift the patient to a medical center for a medical examination; The patient does not have to change his way of life; The patient does not have to change the routine of their daily activities or work; The patient does not need to change the pace and type of food; The patient does not have to stop, or modify any medication; No false negative or false positive results, because for the test specification merely count the ribosomes of colonocytes, which are cells that are malignized in cases of colorectal cancer, or in cases of malignant process.

The advantages of the RIBOGRAMA METHOD over other colon and rectum noninvasive cancer tests are twelve evident factors that by themselves provide a good patient compliance and preference, determinant for health professionals, because there is no false positive or false negative results, since RIBOGRAMA is considering the loads of ribosomes of desquamated cells of colorectal mucosa, which are mixed with stools, not being necessary the colonoscopy to know whether the patient is, or not, in a process of gut malignization.

The project consists of performing a proof of concept during two years, allowing the validation of results with which it is possible to make comparative studies and may be possible to build good and credible values quantifying the levels of free ribosomes, which are responsible for the production of proteins that serve to double the protein content of cells, which, in the states of malignancy do not have the universal self.

With this method it is possible to prevent people from being caught with a colorectal cancer, with invasion of the submucosa because, as can be seen, the increased amount of free ribosomes, above a certain level, defined the proof of concept in the project, to develop the RIBOGRAMA before the occurrence of any polyp, it is easy to know that a patient began to have an important criterion to be included into a risk group, in which even if there is no character of malignant lesion, at a histological level, such as a seemingly benign polyps, there is already a biochemical profile of malignancy. Within the global scientific community, this method utilizes 60

RIBOGRAMA PROJECT a new way to study the biochemical behaviour of the malignization. It will impact on the economy (and will give unparalleled statistical indices) in the countries. That is, before there is any malignant lesion, visible in optical microscopy (pathologic anatomy), before a colonoscopy, it will be possible the doctor to be sure whether there is a process of cellular proliferation, translated by a noticeable and significant excess of free ribosomes in cytoplasm of colonocytes, which is a common characteristic of a malignant tissue, or with a tendency to becomes malignant.

Against this method, the cell/tumor markers, which are proteins, are less sensitive to study the malignancy, since they are elements manufactured in the free ribosomes. It means to say that these (free ribosomes) are the main elements for monitoring the malignant phenotype (upstream) in the chain of cell production/proliferation, since the cell/tumor markers are downstream to the variation curve of concentration of the free ribosomes (RIBOGRAMA curve).

The method RIBOGRAMA allows, in a non-invasive way, with easy adhesion of the population, to measure the quantity of free ribosomes in the colonocytes (which are mixed in the faeces), by studying the pattern of them in the colorectal mucosal cells, being made possible harvest from collecting the stool in the toilet-fit of the bathroom, in the house of each patient that chooses to conduct the test, without to manipulate their own feces.

As is described in the Abstract, attached to this approach, the method gives a concrete numerical result, and is convincing to the patient's household and those with whom he speaks and lives in the day-to-day, making that other people are induced to do it, as with the control of treatable and controllable diseases (diabetes, dyslipidemia, uric acid, for example). The patient just take home a "collector" of feces (whose design is explained in the RIBOGRAMA project), who will buy it in a pharmacy, a chemist, or a shopping center and, after sampling of feces, at home, guided by an illustrated instruction booklet, will deliver the biologic product in a clinical laboratory, which has its technical suitability and accreditation.

The practical consequences of this method are such that there is a marked decrease in spending resulting from the treatment of colorectal cancer, and a decrease of suffering resulting from the significantly lower number of patients with colorectal cancer, because people know when to go to a doctor's appointment, when the curve of his RIBOGRAMA provides information on the early tendency of the colorectal mucosa to have the risk to becomes malignant, which arise as a result of a significant increased and persistent high RIBOGRAMA curve before any colonoscopy. Patients may repeat the RIBOGRAMA test within a short periodicity at low cost, without the risk of being caught with a lesion on the path to malignancy, with great cost savings to the countries and to the community without having to make a blind colonoscopy, with discomfort with its preparation and its realization.

Speaking of costs with the disease, once diagnosed cancer in a stage of which exceeded the submucosa we can speak in multiple charges, as a result of colorectal cancer, which can be listed in the following list: 1.

2.

3.

4.

5.

6.

Costs of operating theater; Charges for intensive care and/or the recovery room of patients operated from the cancer; Charges for hospitalization of the patient as a result of the operation; Charges for chemotherapy and other drugs; Charges to follow up exams; The Social Security System no longer receives input from an inactive citizen and is paying to an incapacitated; 7. Usually the patient will not go alone to a consultation and follow up treatments, taking with him a family member or a friend who wil not work in his workplace, withloss of productivity to the community.

61

RIBOGRAMA PROJECT It would be useful to present a Symposium related to the RIBOGRAMA topic, aimed at biomedical researchers, University teachers, entrepreneurs and investors on the subject, focusing on the multiple aspects related to the phenotypic behavior of a cell in the process of malignant transformation to reveal the potential of the method, which become a universal tool for study and research in oncology and other areas such as the toxicity of substances on the cellular homeostasis in the pharmaceutical and food industry.

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XIII - INTERPRETATION OF THE RIBOGRAMA CURVE To complement the description being made and in order to help better understand the features of this method, accompanying this specification, as an integral part thereof, of a set of drawings, which are an illustrative mode, represent the following:

The figure shows an example of a scheme in which the axes, which represent the two cartesian coordinates. One constructs a curve to represent the change of the quantity of free ribosomes per cell (or per unit volume) in a defined time period, within an average of the exfoliated cells of colorectal mucosa of a patient.

→ Curve that starts at the normal level, and reaching the level increased, which involves care for a prophylactic study on the reasons or causes that determine its appearance. The cells at this level (increased) still have no security features of malignancy, but their increased number of ribosomas continues growing. The cell is transformed into a malignant phenotype (alarm level). At this point it is essential to make a dietary study to survey and identify possible mutations responsible for the uncontrolled growth of ribosome biogenesis in the process of carcinogenesis; → Segment of the curve derived from the anterior (segment a-b), which corresponds to the phenotypic alterations and to the visible light microscopy in which cells present already with phenotypic features of malignancy;

→ Segment of the curve, continuing the anterior segment, which represents the level of signs and symptoms of the CRC disease (clinical level); → This segment of the curve corresponds to a change in direction of the curve a-b that results from preventive action and treatment (medications and special diet);

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RIBOGRAMA PROJECT → corresponds to a curve in which cells grow (multiply or proliferate) in a self-monitoring mechanisms, increased proliferation, as in the case of the seminiferous tubules, or the endometrium. This increased level of ribosome biogenesis the control thereof is within the physiological (non-malignant); → Normal curve;

→ This work is intended to study the item colorectal cancer (CRC), in which ribograma curve is high, so it is not the time to talk about the meaning of it at low level. Still it will be useful to study conditions of degeneration and involution of cells and tissues, either the intestinal mucosa, or other extra-intestinal tissue.

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XIV - RIBOGRAMA â&#x20AC;&#x201C; IS A BIOSENSOR TO MONITOR CARCINOGENESIS RIBOGRAMA is new concept in the modern biomolecular clinical oncology based on the transition from the subjective and qualitative understanding of the processes in the tumor genesis of cells to digital and numerical-suitable way for computer analysis. One place to start, as well, were the published data on 60's and 70's, and research related to the accumulation of free ribosomes under the chemical induction of tumor growth, through cell initiating, with 7,12-dimethylbenz(a)anthracene and promotion caused by 12-Otetradecanoyl-phorbol-13-acetate in the interfollicular area of the back skin of mice. However, their form description received only important literary and scientific results, at that time, but did not allow a quantitative description of the process. According to the trend noted in the cells, were observed characteristics of cancer, with heavy increasing number of free ribosomes. In the series of works, the process of studying cell carcinogenesis is then putted in a new level with the digital definition of pathological processes, taking place with free ribosomes. It becomes one objective visualization that is read out by creating a graphic curve, with numerical values corresponding to the RIBOGRAMA which gives concrete values of the trend to the cancer.

Analyzing the construction of the curve, using statistically derived gradation number free ribosomes one finds abnormal and marked increase in their number, and the transition to the dangerous increase in the cancer process which, at this stage, is a diagnostic tool available in the arsenal of modern clinical oncology previously to the cancer biomarkers and to the pathomorfologic changes in the tissues under study. Patents and copyrights N Âş 200701216 (2) MCT Esp. 02/18/2010 (SPAIN). To calculate the number of free ribosomes it is used the flow cytometry method, after their isolation and labeling with fluorochromes. Giving mathematical direction, it allows the statistical computer processing of the received material, the formation of different groups according to the degree of risk, taking into account various factors such as individual, family, geographic, professional, etc.

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XV - RIBOGRAMA - A NEW METHOD IN THE STUDY OF CARCINOGENESIS – (BIOMOLECULAR CONCEPT AND ITS USE IN CLINICAL ONCOLOGY AND LABORATORY ANALYZE) The novelty of the work: RIBOGRAMA – is a non-invasive laboratory method in the study of the dynamics of the current cancer process, through its screening and monitoring at the cellular level, i.e. even before the appearance of early histological and clinical manifestations in the body. The method is based on the result of the literature in the field of electron microscopy which utilizes the latest Molecular Biology, Biomedical Engineering and Informatics. The study is based on the calculation and statistical analysis of the dynamics of the change in the number (density) of free ribosomes in the cytoplasm of eukaryotic cells, the so-called phenotype of the analyzed organs and systems, and the construction of individual RIBOGRAMA. It is shown that in the cell a very big increase in the biogenesis of free ribosomes (and the simultaneous increase of RNA) indicates the presence of cancer.

Patent and copyright: Nº200701216(2) MCT Esp. 18.02.2010 (SPAIN).

Materials and Methods: The method is based on a retrospective analysis of 206 basic and well-documented scientific observations in the field of electron microscopy of cells with a malignant phenotype and also used a database of literature in PubMed. It is based on detailed changes in malignant tumor cells and tissues. It was developed a method of quantification of free ribosomes, with help of the principle of flow cytometry after isolation and labeling them with fluorochrome staining. With a sequence of records obtained with the numerical values representing the quantity of free ribosomes it is possible to do a mathematical analysis of the dynamics of free ribosomes biogenesis and then to interpret the process of carcinogenesis, by reading the RIBOGRAMA curve. In this way are obtained values and their changes, giving evidence of the existence and nature of a malignant process.

Results: The descriptions of the morphologic aspects of cells in each tissue, under study, were critically analyzed (in the qualitative aspect). In this way, were created six different groups on the search, relatively to the quality content of free ribosomes in cancer cells (in the 1st group- NUMEROUS FREE RIBOSOMES-36 observations; in the 2nd- RICH IN FREE RIBOSOMES-5 observations; in the 3rd-ABUNDANT FREE RIBOSOMES-36 observations; in the 4th- MANY FREE RIBOSOMES -19 observations; in the 5thINCREASED NUMBER OF FREE RIBOSOMES-28 observations; in the 6th- FREE RIBOSOMES-70 observations). Some other terms have been used, which were not so frequent in the groups of 206 cases collected and then have not been highlighted. Of the available scientific evidence, the morphologic view (Electron Microscopy) does not allow an accurate mathematical analysis to identify groups of evidence - based trends in cancer process - which, in the end, did not allow the adoption of the correct tactics of treatment in the clinic. The proposed present quantitative method followed the construction and analysis of individual recording of quantities of free ribosomes, which allows to analyze and predict the dynamics of perspective directions and trends of cell groups, after making a graphic curve RIBOGRAMA. With the construction of individual RIBOGRAMA curve to quantify the abnormal, enlarged and dangerous quantity of free ribosomes - in the temporal aspect – the time latency of each numerical change and, subsequently, the possible transformation and transition to the phase of clinical manifestations (before the change of blood markers and histological changes …), the normalization process or apoptosis, we are monitoring the behaviour of a future cancer cell.

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RIBOGRAMA PROJECT The relative diagnostic value, of the non-invasive methods available today, for example - feces occult blood test (for colorectal cancer) and/or DNA mutations (or any other organ and system) are very time-consuming, with false-negative and false-positive results, and the need of special conditions, equipment and personnel, etc. - colonoscopy (which has a certain percentage of risk) in the study of colon and rectum cancer - or other organs - makes the RIBOGRAMA method much more preferable, because it is painless, highly informative and rigorous in clinical oncology and laboratory research, and also allows us to conduct massive research in thousands of patients, making all things being equal, practical and very economic to the patients and the countries. Tumour markers are proteins, which, usually, are produced by the cancer cells, what means that when we start using tumor markers it is already established a state of cancer in the cells under study.

Furthermore, the method allows to induce and see the change of the RIBOGRAMA curve, under external stimuli to the cell. The dynamic and mathematic accuracy control of the method allows to interpret the success of a treatment with a special diet and proper selection of drugs by quantitative change in the RIBOGRAMA curve. We can analysis and monitor (the so-called secret allocated studied organ) the subsequent construction of guide lines and its analysis in the dynamics of a treatment. It allows the discharge of patients in various groups of degrees of cancer risk, their follow-up and regroup during their treatment. It is possible to monitor large number of observations and groups of patients and make effective analysis of trends, both in conservative and in surgical treatments.

Conclusions: 1. Significant great increase in the accumulation of free ribosomes in the cytoplasm of cells of any tissue may be one of the possible phenotypic characteristics of malignant transformation of these cells; 2. RIBOGRAMA reveals the limits of normal concentration of free ribosomes, to determine their concentration per unit volume. A great increase in their quantities is a sign, at the cellular level, malignant transformation, long before clinical manifestations - the appearance of blood-specific tumor markers, histological transformation of tissues and DNA mutations; 3. Phenotypic expression in the cytoplasm, in a significant great increase in the number of free ribosomes (by levels of concentration) is a sign of tumor growth and may be considered as a prognostic factor in cancer patients; 4. These RIBOGRAMA data confirm that there is a correlation between different numerical content of free ribosomes in the cells and their dependence on the degree of tissue differentiation (morphological and physico-chemical studies); 5. Quantitative analysis of free ribosomes, their dynamic performance in the form of a graphic curve RIBOGRAMA- allows to analyze and predict the trend malignization of each case, the choose of the most appropriate treatment and get the correct orientation and reasoned in the diet and drug therapy in each case.

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XVI – RIBOGRAMA: CONCEPT AND CLINICAL APPLICATIONS IN CANCER THERAPY - (A NEW METHOD TO SURVEY CARCINOGENESIS SCREENING, DIAGNOSIS AND THERAPEUTIC) A. Ferreira-Alemao, MD PhD UNIVERSIDAD COMPLUTENSE DE MADRID

1 - ABSTRACT The purpose of this research, which is was presented as a Doctoral Thesis, at UNIVERSIDAD COMPLUTENSE DE MADRID, with the above title, is the following: - Draft a method of diagnosis, screening and monitoring the cancer disease founded on technical and current concepts of Molecular Biology. This new method allows a diagnosis in a "pre-carcinoma in situ” stage, since it is based in the phenotype of cells, composed of very high quantities of free ribosomes in its the cytoplasm, which can be quantified by means of the of the flow cytometry, after its isolation, and marked with fluorochromes. The repeated records of those quantities of free ribosomes, establishes a graphic curve (RIBOGRAMA) that represents the degree of a malignant tendency, which above a certain level of concentration, allows to say that the cells in a tissue or collection of cells can be considered in a process of developing cancer, before any macroscopic or microscopic viewing. This means, we are facing a new method, on a clear biomolecular level, as shown in the Doctoral Thesis, with advantages (for good progress) on the macroscopy and microscopy. This method exceeds the scope of other non-invasive methods. Thanks to this method, it is possible to have a screening program in which all citizens who present a increased curve of RIBOGRAMA must be mandatory checked with a diagnostic and therapeutic clinical tools, and at the same time, remain under observation and therapy, with proven efficiency in the prevention and reduction of the premalignant tendency injury, representing great savings in the therapeutic inversion of cancer.

2 – INTROIT The RIBOGRAMA METHOD is a real biological biomarker to monitorise the behaviour/phenotype of a community of cells under study/treatment (e. g. breast, colorectal tract, upper digestive tube, lung, prostate, gynecologic tract, etc), before metastatic cancer explosion. If this biomarker is adopted, we can analyze someone's tumor tissue before treatment and see how they respond to medication and follow the rules of personalized treatments.

The RIBOGRAMA METHOD is a strong tool of the chemistry, manufacturing and control of the new (and/or orphan drugs), as well the old medicines, because it is based on the mechanisms of the universal biological macromolecules in the cytoplasm, like the free ribosomes. The RIBOGRAMA METHOD is an in vitro predictive biopharmaceutical tool for drug therapeutics choice, especially in sensivity to cancer cell lines 68

RIBOGRAMA PROJECT and to stem cell cultures (in view to the sensivity of cell upstream machinery to the free ribosomes), since one can read the highness of the RIBOGRAMA curve in front to a certain action of a substance.

The RIBOGRAMA METHOD helps the quickly decision in the choice of the best antiproliferative drug in cancer therapy and the understanding of the behaviour of human induced pluripotent stem cells to the drug action. The goal of RIBOGRAMA is an innovative biomolecular method for very early diagnosis (not invasive) of cancer, which is based in a simple new biomolecular approach, similar for all types of cancer, particularly hematological cancers and cancers affecting the lung, breast, gastrointestinal tract, pancreas, gynecology and urogenital tracts.

It will enhance the life of patients and improve the management of cancer disease, allowing monitoring the efficacy of any cancer treatment and other non-cancer diseases. According our present understanding of the multiple known basic mechanisms of the uncontrolled cell growth and metastasis, there is a growing big number of multiple pathways, from the malignant tumour growth to angiogenesis and metastasis that complicates the design of a transversal method of diagnosis of cancer of eukaryote cells.

There is a very wide source of information on cell signaling pathways (up - and downstream in the cell) which makes the talks in the cellular biomolecular processes complex and difficult to read the global context, such as in signal transduction resources, information on Akt signaling, apoptosis, cancer pathways, caspase cascades and pathways, the cell cycle, cell death pathway, cell stress, cyclic nucleotides, cytoskeleton, extracellular matrix signaling, G proteins, gene regulation, growth factors, hormones, immune cell signaling, ion channels, lipids, multi-drug resistance, neurobiology, neurotransmission, nitric oxide, phosphorylation, receptor signaling and receptor signal pathways, secondary messengers and signaling molecules.

The number, the cross-talk and complexity of the signaling ways in the function of the cellular biomolecular network naturally become an obstacle to design a transversal method to diagnose a hyperproliferative state of a community of cells. With the RIBOGRAMA METHOD it is possible to design a curve of quantities of free ribossomas in the cytoplasm of a community of cells under study, allowing in advance the monitoring of its biomolecular response, in a global and integrated fashion, allowing to know if we are in a state of hyperproliferation or in a diminishing of the state of proliferation.

Defects in cell-signaling pathways allow cancer cells to alter their normal programmes of proliferation, transcription, growth, migration, differentiation and death. Tumour formation and progression occur through a range of defects that develop both within and outside the cancer cell. Many different anticancer agents have therefore been developed to target proteins that act in many of these pathways. These very important aspects about the cell behavior can`t give a monitoring quantitative information like the RIBOGRAMA.

3 - A PERSPECTIVE ON THE BIOLOGY OF MALIGNANT CELLS Several lines of evidence, some well established and recent, say emphatically the idea that RNA content is high in cancer cells and genetic events that lead to cancer are often linked, directly or indirectly, to the ribosome biogenesis (178.179).

On the other hand, the inverse problem is checked in the cases of "idiopathic ineffective anemiaâ&#x20AC;? (IIE Ineffective erythropoiesis idiopathic), where there are insufficient rates of cell proliferation, and patients have erythroblasts containing only 70% of normal levels rRNA (185). Then, in a situation of excessive cell proliferation (aggressive cancers) rRNA levels are amplified, whereas in a situation of insufficient cell proliferation (certain anemias marrow hypoplasia) rRNA levels are depressed.

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RIBOGRAMA PROJECT One have been developed oncobiologic investigations which have shown that the expression of the alterations of rDNA (ribosomal deoxyribonucleic acid) and ribosomal protein genes are associated with the development of tumors and cytological studies show that the nucleolus of cells cancer are increased because they have an increased transcriptional activity, representing predictive aspects of cell proliferation rate and prognosis of patients (186.187).

These researchers have shown that cancer cells with high expression of these ribosomal proteins have higher content of ribosomes. The genetic alterations associated with cancer development involve, very often, changes in signaling pathways that lead to the rDNA effect (186.187). The scenario described above reiterates what is known by biologists cancer: ribosome biogenesis and tumorigenesis are closely linked.

4 - ACCUMULATION OF RIBOSOMES IN THE PROCESS OF ONCOGENESIS In the decades of 60 and 70 were published studies on free ribosomes and ribosomes bound to membranes (201,202) and its accumulation during the induction of growth in many organs and tissues (191-194). In the sequence of these investigations are published studies that have attempted to quantify the accumulation of ribosomes in interfollicular areas of the dorsal skin of mice during chemically induced neoplastic growth in two phases, initiation by 7,12-dimethylbenz (a)anthracene and promotion caused by 12-O-tetradecanoyl-phorbol13-acetate (1073).

The epidermis is a surface epithelium and like other epithelia, which are in direct contact with the external environment, such as the linings of the respiratory and gastrointestinal tracts, show a high incidence of cancer.

Thus, the epidermis has served as a useful model to establish a comprehensive development of the role of ribosome accumulation during neoplastic growth (207).

5 - POTENTIAL BIOMEDICAL APPLICATIONS RESULTING FROM THE COUNT OF RIBOSOMES The preparation of ribosomes, depending on the tissue under study, can be done through some adaptive modifications, published in previous studies (196-200). The preparation of ribosomes can be used for many purposes, for example, to study the growth and development of tissues induced by hormone action (1071), the study of gene expression, including synthesis of ribosomal proteins which are translational control (204-205), the study of "assembling" of the ribosome (1072), study of differential expression of ribosomal proteins of the mucosa of the colon and rectum normal and neoplastic (206), among many other potential, and most likely, in nonmalignant diseases.

The identification of changes in the structure of the cytoplasm, such as the sharp increase in the number of ribosomes (quantitative method) accumulated in the cytoplasm of a community of cells of a given tissue may be one of the phenotypic expression malignancy of the fine structure of the transformed cells, which may be essential to characterize the evolution of cell behavior.

In this sense, for example, could study graphic profile of changes in the number of ribosomes per cell (or per unit volume) in a defined time period, within an average of the exfoliated cells of colorectal mucosa of a patient, which will form a standard curve/ average number of ribosomes per unit volume RIBOGRAMA in relation to the cells lining the colon and rectum, isolated and separated from the feces.

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RIBOGRAMA PROJECT Like many fields of life sciences, biology of cancer is an exponentially growing field and highly complex, including work that has a range from molecular biology to epidemiology oncogenes environment. Survival rates for various cancers, once clinically manifest, have shown modest improvement in recent decades.

There is a strong motivation to integrate different fields of knowledge in cancer biology and introduce a new conceptual and theoretical framework that can improve the understanding of tumor dynamics researchers so, to develop better therapeutic measures. It will be important the production of mechanistically predictive models based on tumor dynamics that can abstract the meaning of the relatively advanced biological details to oncogenesis and tumor progression. While it is true that cancer is a multifaceted disease with a variety of close "triggers" in different tissues and different patients, there is also a strong possibility that cancers share a central feature originating from a common cellular machinery which the cells depend for their proliferation (177).

The most visible aspect of aggressive malignancies is increased cell proliferation, which has at its core a marked increase in protein synthesis. In the process of normal mitogenic response, there is a transient and cyclical increase in the rate of general protein synthesis. The overall increase in protein synthesis is a necessary phenomenon observed controlled before cell division, leading to duplication of content and the increase in size before the normal mitosis. Thus, the average size of the cells is maintained during physiological proliferative response.

One of the key mechanisms of loss of control of protein synthesis in transformed cells is the inability to decrease the number of ribosomes that is correlated with cell proliferation in fresh culture media without added growth factors to serum (156). Many researchers have observed in tissue cultures, differences in growth properties between normal cells and their malignant counterparts, one of which points to the failure of these to show a cyclic variation of cellular and biochemical parameters through the cell cycle or growth cycle (159-162).

Previously, other researchers have given attention to changes that occur in the rate of protein synthesis and function of machines "translational" of the cell, relative to the cell cycle, once such changes are necessary transitions growth and multiplication in normal conditions.

6 - THE NUCLEOLUS In this sense, it makes sense to talk about the fact that many pathologists during the routine examination of biopsy material evaluation, often pay attention to the nucleoli (the sites where rRNA is synthesized and ribosomes are "assembled"), as in malignant neoplasms, nucleoli are altered in some way relative to the number and size. Thus, the association of nucleoli altered in neoplastic disturbances has been referred to research from more than fifty years ago (157). The nucleolus is a functional, well-defined structural unit in the interphase cell in which ribosomal genes are located and where ribosomal RNA synthesis (163) occurs.

Among other functions, the nucleolus, which is a key organelle, coordinates the synthesis and assembly of ribosomal units.

The production of ribosomes is an important metabolic activity and thus the role of the nucleolus is closely linked to cell growth and recent data suggest that the nucleolus also plays an important role in cell cycle regulation, senescence and stress responses (188).

The ribosome biogenesis involves the synthesis of rRNA, maturation and assembly of RNA and ribosomal proteins in ribosomal subunits, small and large. This process is regulated through the cell cycle, primarily at the level of rRNA synthesis (189). RDNA transcription reaches its peak during the S phase and G2, is interrupted when the cell enters mitosis and reactivated when cells exit mitosis (190).

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RIBOGRAMA PROJECT In the last ten years have acquired significant knowledge about the significance of changes in the nucleolus in tumor pathology, through studies of changes in distribution of a group of nucleolar proteins, proteins AgNOR (Argyrophilic Nucleolar region Organiser) in tissues malignant. These proteins are required for ribosome biogenesis and are selectively stained by silver methods (164). The two most important proteins nucleolin and protein B23 are involved in rRNA synthesis and processing. (165).

On studies of tumor biopsy fragments for in situ observation cytohistological and isolated proteins, it is verified that the amount of AgNOR proteins increases progressively when resting cells enter the mitotic cycle from G1 to the end of S phase (166 - 168). Also, there is evidence that the amount of AgNOR proteins is directly related to the rapidity of cell proliferation in cell lines established human cancer (169-171).

The NOR (Nucleolar Organiser region) are sites of rRNA transcription, modification post-transcipcional of RNA transcripts and its "assembly" in functional ribosomes. The number of NORs expressed in a tissue is related to the rate of cell proliferation, with differentiation and neoplastic transformation. This has been used to demonstrate the potential malignancy and to assess prognosis and malignant aggression (173-175).

Quiescent hematopoietic cells such as the not activated T and B lymphocytes, and pluripotent stem cells, are small with low content of protein and RNA than their counterparts in the state of proliferation. During the transition from G0 to G1 there is a mandatory increase in cell mass and the number of ribosomes (1069) and an increase in the rate of protein synthesis, due in part to increased initiation factor eIF-4E (1070).

In skin tumors in mice induced by the application of tumor promoters, the RNA:DNA relation and RNA content (percentage of dry mass contributed by RNA) were 2 to 3 times higher than in normal tissues (180). In several types of leukemia, the cell RNA content was strongly correlated with accelerated cell growth kinetics and the prognosis of the patient (181).

In a study of gynecologic cancers in which the neoplastic tissues were compared with their normal counterparts, the content of DNA and RNA content in the neoplastic tissues were increased 1.6 and 2.4 times, respectively (182). Similarly, cellular RNA content was increased by a factor of 1.4 in neuroblastoma cells myctransfected cells relative to normal (183). In another study of breast cancer, the test of cell DNA content was normal, but the cellular RNA content was well correlated with tumor grade, histological type, with the hormonal status and patient survival (184).

(The bibliography of this INTROIT is in the Doctoral Thesis, whose entire content is linked to this RIBOGRAMA PROJECT)

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XVII - INTRODUCTION TO THE STUDY OF THE VERY EARLY DIAGNOSIS (PRE-IN SITU STAGE) OF COLORECTAL CANCER (CRC) The importance of an increasingly early diagnosis is unquestionable, because it permits a less aggressive and more effective as well as increased chance of cure. As better and more is the people informed about the cancer disease and its inherent conditions, the better is the collaboration and decision at its therapeutic level.

The potential of cure for colorectal cancer (CRC), after surgical resection, has not evolved in the past 50 years, despite significant improvement of methods of surgical technique and perioperative care. Formal and classic tools of prevention and treatment depend on invasive direct visualization techniques, such as flexible sigmoidoscopy, colonoscopy or barium enema double-contrast, which have failed themselves to lower their incidence and their cause of death.

Colorectal cancer is the second most common visceral malignancy in the United States and around the world eating food and with Western lifestyles, and if diagnosed before the onset of symptoms or clinical signs, patients are more than twice the hypothesis of having limited disease (Dukes' A and B), with better prognosis and potential for cure.

The CRC is a leading cause of cancer death in the world with a Western lifestyle, but is within the best curable cancers when identified at an early stage. It has a long premalignant phase and a slow progression from the stage of disease confined to the wall of the organ, to the stages of local invasion and distant metastatic disease. Therefore, there is ample opportunity to identify patients at a curable stage with the completion of large screenings. Colorectal cancer (CRC) is a disease that can be prevented in advance when colorectal polyps are removed, or the disease is highly curable when detected in its initial state.

It is important to have screening and diagnostic tests to examine the entire colon and rectum, because the cancer usually does not exhibit signs and symptoms in its early stage. Screening the CRC is made in individuals without any signs or symptoms that might indicate a suspicion of cancer. To prevent the CRC is crucial to understand its causation and pathogenesis, which is a prerequisite for effective action. Causality can be established by combining the epidemiology (a key tool to identify major risk factors) to the investigation of the mechanisms of carcinogenesis.

There is need for epidemiological health policy, which has, in part, to be addressed with hereditary cancer syndromes and genetic issues and understanding bimolecular machinery underlying the pathogenesis of the onset and development of the mutant phenotype to the onset of cancer with signs and symptoms, in which the healing is already a mystery and uncertainty.

Today one cannot wait for the signs and symptoms, but yes one must go to meet the advent of the causes of cancer. The strategy is not to expect, but progress on the way to meet and combat the causes and find the cancer in a early phase of healing. Everything must be cast in the "pre-in situ", which is the best time to speak to cure cancer.

The CRC has a long premalignant phase and a slow progression from the stage of disease confined to the wall of the organ to the stages of local invasion and distant metastatic disease. Therefore, there is ample 73

RIBOGRAMA PROJECT opportunity to identify patients at a curable stage with the completion of large screenings. Currently, methods of approach are not yet specific, such as the investigation of occult blood in stool, or those that rely on invasive techniques for direct visualization, such as flexible sigmoidoscopy, colonoscopy or barium enema. Recent advances in clinical chemistry and molecular biology led to the emergence of new techniques for noninvasive approach, based on the biology underlying CRC. This approach identified mutations that are known to be associated with CRC in the DNA that is shed in feces for malignant and premalignant lesions of colon.

Despite many studies over the past 10 years, the impact of many dietary factors in colorectal carcinogenesis is still not resolved. Colorectal cancer trends in the United States are significantly different from those of Europe.

In the United States, the incidence has increased slightly since 1971, while in late 1990 mortality has declined about 50% since 1950. These trends are consistent with survival rates at five years, improving from 20% in early 1970 to almost 45% in mid-1990.

In Europe, there has been generally decline in the mortality of the women than in men, which may be due to increased use of contraceptives and hormone replacement therapy. Mortality has fallen recently in many European Union states, but is increasing in Greece, Portugal and Spain. Despite the accumulated evidence, including randomized controlled studies on the effectiveness of the investigation of occult blood in stools, which confirms the existence of a reduction in colorectal cancer mortality in a program applied to a certain population, many people in developed countries have not had been submitted to any screening.

One-fifth of the series of "Annual Reports to the Nation the Status of Cancer" highlights data from the four most common cancers - lung, female breast, prostate and colorectal - which in total represent more than half of cases of cancer and cancer deaths in the United States. These four cancers are equally important in most of Europe. There are over 20 years, Doll and Peto using data from studies done in 1970 and before, concluded that mortality data were generally more credible than the incidence data.

Still, data from cancer registries in the world famous office epidemiological data SEER (Surveillance, Epidemiology and End Results) of the National Program of Cancer Registries (NPCR), which follows the high quality criteria released by the "North American Association of Central Cancer Registriesâ&#x20AC;? (NAACCR), and a large number of records worldwide, follow the most high quality standards published by the IARC (International Association of Cancer Registries), making it very believable. The mortality data were never exempt from criticism or trend.

The trends in incidence and survival of cancer registries provide an additional comment on the complex problems of cancer control. In the past 30 years the survival of almost all cancers has improved genuinely, at times dramatically. The implications are that the trends in incidence and mortality have diverged. If this seems true, then this general agreement on the results of two systems that are largely independent record induce cancer and death greater confidence in each of them. But no incidence, survival, or mortality is perfect and none is adequate in itself.

In Europe, cancer registry systems of people, from many years, with national coverage in each country (often regionally organized), and follow-up cases, are virtually complete in the Nordic countries (Denmark, Finland, Iceland, Norway and Sweden), the United Kingdom and many countries of the Baltic and Central Europe, former Soviet Union. Coverage in other parts of Europe, like France, Germany, Italy, Portugal and Spain, is, however relatively poor. In this context, the establishment of a rigorous cancer registry is an important route in the direction of cancer control.

So far, the non-invasive diagnostic methods utilized as the investigation of occult blood in the stool, have not been specific. Published trials based on the method for blood (in search of the iron molecule) in the stool, made twice a year, have shown a low of 15% -18% reduction in mortality, while the method gives a lower immune 30%, being more specific and safer. The positive test for fecal occult blood has a high percentage of 74

RIBOGRAMA PROJECT false positives and false negatives are not negligible. When a tumor is bleeding, this means that the mucosal barrier has already been destroyed by erosion/invasion of the submucosa, with their blood and lymph vessels, ensuring no certainty as to the metastization and lymph node involvement, or no tumor Tis is a / 0.

The development of noninvasive tests, derived from knowledge of basic tumor biology, shows the promise of improved targeted barriers to colonoscopy, an examination of screening and diagnosis of CRC. We are aware of human DNA mutations in stool, which are associated with the CRC and premalignant colorectal polyps.

The knowledge of this fact has led to the appearance of a basic methodology (CRC screening) studying mutations in the DNA of cells shed from the colorectal mucosa. The science behind the study of DNA mutations is sophisticated. When neoplasms are developed, cells and fragments are scaly and released in the feces in the intestinal lumen, leading the stool DNA comprising the sample for testing.

The testing of mutations and feces have false positive and false negative, with several large amplitude, say the authors. Mutations in the trials and feces may belong to other organ (respiratory tree, gastrointestinal tract, upstream of colorect, etc), not allowing the assurance of the location of possible tumors in the alimentary tract.

In addition, mutations in the tests (mutations of DNA in feces) may not be mutant phenotypes, which contribute to the great percentages of false positives.

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XVIII - PROCEDURES FOR PREPARATION AND COUNTING FREE RIBOSOMES (TECHNICAL PHASES FOR CONCRETIZATION OF THE RIBOGRAMA METHOD) For the counting of free ribosomes in the unit volume under study will employ the following techniques and/or phases: 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

Obtention of a sample of cells or tissue to study Separation of the cells to study Break of tissues and cells Homogenization Subcellular fractionation Centrifugation Sedimentation Differential centrifugation Equilibrium sedimentation Preparation of free ribosomes and tagging with fluorochromes 11. Counting of free ribosomes with the use of flow cytometry or with reverse-transcriptase qPCR or RT-qPCR.

1 - THE ESSENCE OF THE METHOD In the framework of the mentioned figures are designed the two axes that represent the two cartesian coordinates (horizontal axis and vertical axis) with which one built a graphical curve. This curve is characterized by two variables about the free ribosomes of cytological community of cells under study (the epithelial cells of colorectal mucosa or colonocyte). These variables are: "quantityâ&#x20AC;? (number) average of ribosome-free in the cytoplasm of each cell and "time" over which is observed the behavior of certain variations of the "quantityâ&#x20AC;? (number) mean free ribosomes.

There will need to raise experimentally graphic profiles of numerical values (mean quantity/number of free ribosomes) that can be set as normal, from which one can draw a curve-middle-register. Thus, it is possible to make comparative studies of large population groups, which will be possible to see multiple potential trends of malignant disease (by raising the curve) in a community of cells in a tissue under investigation. In studies correlating electron microscopy and biochemical data (usually quantitative), the morphological (electron microscopy) is more limited and dependent of descriptions based on subjective criteria.

This criterion (electron microscopy approach) is subjective because it does not allow a statistical measurement of data, with mathematical rigor, preventing a parametric correlation with morphological data for biochemical 76

RIBOGRAMA PROJECT data. The outline of the figure is a record of the quantitative assessment of free ribosomes in the static and dynamic sense, as information collected from subcellular fractions, which can be correlated with other parameters of quantitative cell biological measurement (eg, some tumor markers, eg CEA.).

From the knowledge of the range of values " mean quantity (number) of free ribosomes" considered within the normal range (range A) one may consider other intervals in the outline of the figure are represented by B level increased, C - level alarm, D - clinical level, A -low level and 0 absent. In the horizontal axis is inserted multiple periods of time, for example, three months, (1, 2, 3,........,10) starting from the beginning of the monitoring records of the values of "quantity (number) free ribosomes average " of the cells under study.

Descriptions of measurements of the cells with phenotypic characteristics of malignancy vary by greater or lesser degree of differentiation of its texture or morphology, but these descriptions are subjective, depending on the observer who does research. The same happens when free ribosomes are descripted under Electrom Microscope. Free ribosomes can be counted mathematically, or its quantity can be measurable and comparable over time, as can be done according the RIBOGRAMA METHOD. The free ribosomes can be quantified by counting them, using flow cytometry techniques, after isolation techniques, used for log time in cell biology techniques. Thus, the procedure for screening and early diagnosis of cancer or its monitorization treatment, which advocates the present invention is based on monitoring the quantitative profile graphic of the change of number of ribosomes per cell (or per unit volume) in a defined period of time, within an average of the cells, isolated and separated, whose records will form a sequential pattern/average curve of the quantity number of free ribosomes per unit volume, after an adequate isolation of free ribosomes, using a certain standard techniques.

The application of molecular biology techniques to isolated cells of any organ profoundly increases the sensitivity of the detection of cancer or its tendency to malignant transformation, enabling its use in the diagnosis and screening. RIBOGRAMA curve has a mathematical basis (that may be reproducible in equivalent circumstances), which corresponds to a non-subjective language; Analysis of trends and changes of the quantity of free ribosomes found, corresponding to that curve, will be a reliable indicator of the degree of development and propensity for malignancy; The free ribosomes can be quantified by counting them, using flow cytometry techniques, after isolation techniques, used for log time in cell biology techniques. Thus, the procedure for screening and early diagnosis of cancer or its monitorization treatment, which advocates the present invention is based on monitoring the quantitative profile graphic of the change of number of ribosomes per cell (or per unit volume) in a defined period of time, within an average of the cells, isolated and separated, whose records will form a sequential pattern/average curve of the quantity number of free ribosomes per unit volume, after an adequate isolation of free ribosomes, using a certain standard techniques.

The RIBOGRAMA phenotype corresponds to a quantitative basis of free ribosomes, that can provide quantitative information on levels of risk and significance, prior to the "open" malignancy, and information on the risk profile and the heredofamiliar profile, food, dietary and lifestyle habits; With the RIBOGRAMA METHOD there are different levels of amounts of free ribosomes on grounds of risk and significance, allowing time to take preventive and therapeutic attitudes against a tendency of malignancy.

In the count of free ribosomes, one is measuring the real uncontrolled increase in the synthesis of proteins, because a tumor depends crucially on the excessive production of proteins in the growth process, that in the cell can only be done by free ribosomes;

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RIBOGRAMA PROJECT Regarding the implications of further development and implementation of the RIBOGRAMA METHOD, this project intended to bring together the material conditions, scientific and human resources for its implementation and execution.

Should be established experimentally graphic profiles of average values ("quantities") of free ribosomes, which will be considered as normal, so that from which one can draw an average curve standard, like the type exemplified in figure of RIBOGRAMA The normal curve of RIBOGRAMA must obey to the limits of a range, within an area considered normal, based on the results of measurements of concentrations of ribosomes from cells of a certain organ to be studied of healthy young adults, aged from twenty to fifty.

After a well-defined limit of normal concentrations of free ribosomes it is possible to do comparative studies, which will create levels of trends of malignant disease, from following certain values of the results found in the course of the time, which are out of the limits of the curve.

The method is designed to work with biologic products of the human body. In the execution of the method there is no procedure on the human body, and technico-scientific conduct will obey to the anonymity and confidentiality under medical and ethical rules.

To define the boundaries of the study in the target population one must be borne in mind that colorectal cancer (CCR) sporadic (which does not depend on heredofamiliar characteristics) has essentially its emergence from the age of fifty.

Thus, for this prospective study, to build the graphical curve of normal RIBOGRAMA and segments of the curve outside the normal limits, there will be four groups of people/patients, using the following criteria: GROUP I - People/patients aged to a maximum of fifty years, considered to be clinically healthy, not having abnormal bowel habits history of defecation, with no episode of loss of macroscopic blood in stools, in the last three years, of whom to obtain stool samples; GROUP II - Faecal samples from persons submitted to rectosigmoidoscopy/colonoscopy with negative results for macroscopic polyps o polypoid formations; GROUP III - Stool samples and biopsy material of persons under rectosigmoidoscopy/ colonoscopy, with positive results for macroscopic polyps, polypoid formations, or suspected cancer; GROUP IV - Stool samples and biopsy material of pieces of people undergoing surgery for colorectal cancer surgery.

The number of samples of health subjects, without complaints or clinical symptoms attributable to the tract of the colon and rectum, for calibration of the normal curve, should be in the order of 500. One obtains a sample from every healthy person selected, for the calibration study of the normal curve, at intervals of three months, which means that for each person are obtained four samples each year. The study for the calibration run for at least two years, which means that, will get eight stool samples from each person in a period of two years.

Thus, it will take 150 to 200 volunteers, in the condition described in GROUP I. People, in the conditions described will be selected from public and/or private or health services, and are submitted to colonoscopies and/or rectosigmoidoscopies, as well as cases of colon and rectal surgery in relation to GROUP IV. From it results a practical and economic way to the community, with easy access to the implementation of the method, for the patients from the perspective of diagnosis and follow up, and general population screening perspective, and what this means in saving lives, when contemplating the incidence of this cancer in a population with their lifestyle and transgenerational eating habits.

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RIBOGRAMA PROJECT The method emphasizes the aspect of accuracy, allowing the temporization and quantification of the ongoing carcinogenesis process in any patient, at random. These are the essential aspects to have in mind so that one can proceed to the PROOF OF CONCEPT of this new method. The method reveals a very simple, practical and economic task to control the cancer disease and a way through which the community will have an easy and safe access. The implementation of the method, to be executed in the daily clinical practice, from the perspective of both diagnosis and follow up and general population screening perspective, means saving thousands lives, when contemplating the incidence of this cancer in a population with their transgenerational lifestyle and eating habits.

For the physician the method becomes a tool for monitoring the potential of oncogenesis of any tissue under study, in that it lets users check the outcome risk quantified of the malignance of colorectal mucosal cells, allowing to intervene in the course of the phenotypic behavior of colonocytes, by utilization of medication, that already exist for other diseases (aspirin and nonsteroidal anti-inflammatory) and a careful selection of certain types of foods, both in how they exist in nature and how they are cocked by people, according lifestyles.

All this will be like using the same methodology as the procedures that medicine employs in the treatment of any disease that has a quantitative metabolic component, or other (gout, dyslipidemia, diabetes, hypertension, etc.), whose scientific laboratorial control depends on the quantitative method monitoring, orientation of the medication, guidance on choice of food and patient adherence to the principles and advice conveyed by her physician, according the values of a quantitative test. It can be said that we are faced with an eminently practical and economical method for the patient and for the doctor which lets face the cancerous disease of the colon and rectum as a chronic illness that can be controlled in a large scale, without recourse invasive and blind procedures, unless the patient arrives to the doctor with already formed polyps.

There is the practical force of this methodology that allows placing patients with high curves of RIBOGRAMA in high risk groups, which then have formal indication to be studied and treated endoscopically. There are medical articles published that talk about the disappearance of polyps with treatment based on diet and prescription medications mentioned above. With this method it is possible to lower significantly the number of deaths and morbidity from colorectal cancer in the world with Western food habits, if sanitary measures are adopted for screening and diagnosis with this simple method, because the degree of public support will be quite high, given sum of the following "factors nice": 1.

2.

3.

No invasive act; No need for the patient, to handle their own feces during collection; No fecal odor nuisance since the fecal collector is industrially designed accordingly (see the design of the colector in the final part of this proposal); 4.

No bowel preparation is needed; 5.

No need for cleansing enemas; 6.

It is not necessary to use cathartics; 7.

It is not necessary to shift the patient to a medical appointment or to a medical center; 8.

The patient does not have to change their way of life; 9.

The patient does not have to change the routine of their daily activities or work; 10. The patient has no need to change the habits and type of food; 11. The patient does not have to stop, or modify any medication; 12. The method does not give false negatives or false positives, because the test specification is limited to counting the ribosomes of colonocytes, which are the targets to be studied in cases of colorectal cancer, or in cases under malignization process, meaning that the test is 100% specific, 100% reliable and universal, for all tissues, once the cells to study are separated.

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RIBOGRAMA PROJECT The advantages of RIBOGRAMA METHOD over other noninvasive tests of cancer of the colon and rectum are the evident twelve "factors nice" that by themselves provide a good patient compliance and a preference for determining the health professionals, because there are no false positives or false negatives, since they are considering charges of ribosomes of cells isolated from the colorectal mucosa.

The project consists of performing a PROOF OF CONCEPT, during at least for two years, which will allow the validation of results, with which it is possible to do comparative studies and thus to be able to take as good and credible figures to quantify the levels of FREE RIBOSOMES, which are responsible for the manufacture of proteins destined to the internal economy of the cell, which serve to double its protein content in the states of malignancy, in which its multiplication is out of control. With this method one can prevent people from being surprised by a colorectal cancer with sub mucosal invasion, because, as is visible, the increased amount of ribosomes, above a certain level, defined in the proof of concept to develop the project before the appearance of any polyps, it is easy to know that a patient has acquired an important criterion to be included in a risk group, in which although there is no risk of malignant lesion with the histological level, such as a polyp apparently benign, there is already a biochemical profile of malignancy.

This method is unique within the world scientific community, and will impact on the economy of the countries.

That is, before there is any malignant lesion visible in optical microscopy (anatomy), before a colonoscopy, your doctor can be sure whether there is a process of very increased cellular proliferation, translated by an excess of free ribosomes in the cytoplasm of colonocytes, which is common feature in malignant tissue, or a tendency to malignancy.

In opposition to this method, the cell/tumor markers, which are proteins, are less sensitive to the study of malignancy, since they are elements (in the cell machinery of protein synthesis) located downstream for the free ribosomes, which is equivalent to say that free ribosomes are the phenotypic elements monitoring the malignancy upstream to tumor markers in the chain of cell proliferation.

This means that cellular/tumor markers in his appearance are downstream to temporal variations of the concentration curve of ribosomes (RIBOGRAMA CURVE). In other words, monitoring the behavior of tumor cells with cell markers is later, or the same as saying that there are now very likely malignant cell dissemination when utilizing the values of cell/tumor markers to monitoring cell behavior, wanting this point mean that the phenomenon of malignant growing takes place downstream (in the cellular machinery for protein building) to the changes in the rising of RIBOGRAMA CURVE.

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2 - BIOMOLECULAR BASE OF THE CELLULAR MACHINERY

The Ribosome as a common element in the biomolecular process of the cells

3 - RIBOGRAMA versus TUMOR MARKERS IN THE DIAGNOSIS AND MONITORING ACCURACY IN CLINICAL PRACTICE In the figure above can be regarded as the path biomolecular located between the initial position of the DNA (where are the genes, which are all more than sixty thousand, but of whom know only slightly more than three hundreds with known functions, some poorly studied) and the final position, which are proteins. Genes work in "concert" with each other, with the result that the uncertain variable functions which might be attributed not allow safety to being assigned as a guaranteed specific and unique. And it remains to determine most of the functions of genes; it is unreliable to make a diagnosis based on some known genes, either for malignant diseases, or for non-malignant diseases, since it is much more what you do not know than what already known.

Having regard to the variable in time, to monitor any parameters of a cellular community, considering the upstream position of ribosomes, on the basis of biomolecular cellular machinery, from the outset there is countless variables that exist in the nature of the genome (some known and others unknown) which do not allow a graphical / mathematical, rather than what is achieved with the quantification of free ribosomes (introducing the concept of RIBOGRAMA). On the other hand, in a position downstream of the ribosomes, the proteins that may be considered markers of a certain line of cancerous cells are the gene products of a cell line; do not allow a very early diagnosis, which does not happen with the slope of the curve the RIBOGRAMA, which allows you to monitor your ascent curve of RIBOGRAMA. The RIBOGRAMA is, for those reasons, a strong, safe and convenient diagnostic tool, to screen, monitoring and prognosis in vitro, in clinical practice and bioanalytical studies. Tumour markers are, usually, proteins, that usually, are produced by the cancer cells. Some tumour markers are specific for one type of cancer, while others are seen in several cancer types. Many of the well-known markers are seen in non-cancerous conditions as well as cancer. Tumor Markers are biochemical substances elaborated by tumor cells either due to the cause or effect of malignant process. These markers can be normal endogenous products that are 81

RIBOGRAMA PROJECT produced at a greater rate in cancer cells or the products of newly switched on genes that remained quiescent in the normal cells. Consequently, they cannot be used to diagnose cancer.

There are only a handful of well-established tumour markers that are being routinely used by physicians. Many other potential markers are still being researched. Some marker tests cause great excitement when they are first discovered but, upon further investigation, prove to be no more useful than markers already in use. The goal is to be able to screen for and diagnose cancer early, when it is the most treatable and before it has had a chance to grow and spread. So far, no tumour marker has gained acceptance in the medical practice as a general screen, including the Prostate Specific Antigen (PSA). The markers are either not specific enough (too many false positives, leading to expensive and unnecessary follow-up testing) or they are not elevated early enough in the disease process.

In 1968 the World Health Organization recommended ten principles to be followed when countries consider developing national screening programmes. The essence of these is that the disease should be important, well understood and be able to be recognized and tested for at an early stage. Medical support and treatment must be available and be more beneficial if given at an early stage. The health benefits must be greater than any harm done by the screening process which itself must be cost effective. Some people are at a higher risk for particular cancers because they have inherited a genetic mutation. While not considered tumour makers, there are tests that look for these mutations in order to estimate the risk of developing a particular type of cancer.

BRCA1 and BRCA2 are examples of gene mutations related to an inherited risk of breast cancer and ovarian cancer.

Screening for early diagnosis has also led to lower mortality for diseases such as breast cancer and cervical cancer. Many malignancies, however, are still diagnosed after the metastatic process has already started, indicating a poor prognosis. Tumour markers are usually proteins associated with a malignancy and might be clinically usable in patients with cancer. A tumour marker can be detected in a solid tumour, in circulating tumour cells in peripheral blood, in lymph nodes, in bone marrow, or in other body fluids (ascites, urine, and stool).

A tumour marker may be used to define a particular disease entity, in which case it may be used for diagnosis.

Tumor markers comprise a wide spectrum of biomacromolecules synthesized in excess concentration by a wide variety of neoplastic cells. The markers could be endogenous products of highly active metabolic malignant cells or the products of newly switched on genes, which remained unexpressed in early life or newly acquired antigens at cellular and sub-cellular levels. The appearance of tumor marker and their concentration are related to the genesis and growth of malignant tumors in patients. An ideal tumor marker should be highly sensitive, specific, and reliable with high prognostic value, organ specificity and it should correlate with tumor stages.

An ideal tumor marker should be highly sensitive, specific, and reliable with high prognostic value, organ specificity and it should correlate with tumor stages. However, none of the tumor markers reported to date has all these characteristics. In spite of these limitations, many tumor markers have shown excellent clinical relevance in monitoring efficacy of different modes of therapies during entire course of illness in cancer patients. Additionally, determination of markers also helps in early detection of cancer recurrence and in prognostication.

A tumor marker produced by the tumor and, when present in significant amounts, indicates the presence of a cancer. They may be present as intracellular substances in tissues or may be released into the circulation and appear in serum. Continuing search for suitable tumor markers in serum, tissue and body fluids during neoplastic process is of clinical value in the management of patients with various malignancies. The spectrum of biochemical tumor markers reported to date is very wide.

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While not considered tumour makers, there are tests that look for these mutations in order to estimate the risk of developing a particular type of cancer. BRCA1 and BRCA2 are examples of gene mutations related to an inherited risk of breast cancer and ovarian cancer. Screening for early diagnosis has also led to lower mortality for diseases such as breast cancer and cervical cancer. Many malignancies, however, are still diagnosed after the metastatic process has already started, indicating a poor prognosis. Tumour markers are usually proteins associated with a malignancy and might be clinically usable in patients with cancer. A tumour marker can be detected in a solid tumour, in circulating tumour cells in peripheral blood, in lymph nodes, in bone marrow, or in other body fluids (ascites, urine, and stool).

However, none of the tumor markers reported to date has all these characteristics. In spite of these limitations, many tumor markers have shown excellent clinical relevance in monitoring efficacy of different modes of therapies during entire course of illness in cancer patients. Additionally, determination of markers also helps in early detection of cancer recurrence and in prognostication. A tumor marker produced by the tumor and, when present in significant amounts, indicates the presence of a cancer. They may be present as intracellular substances in tissues or may be released into the circulation and appear in serum. Continuing search for suitable tumor markers in serum, tissue and body fluids during neoplastic process is of clinical value in the management of patients with various malignancies. The spectrum of biochemical tumor markers reported to date is very wide.

4 - PURPOSE AND POTENTIAL OF THE METHOD The colorectal cancer represents one of the biggest incidences of human cancer in the western type of life and cocked food. This research project has strong potential in public health, medical and hospital practice, clinical and laboratory research, food industry, veterinary, oncologic research, pharmaceutical industry and clinical trials research, ecologic systems, study of nature pollution, etc, with great medical and financial impact, firstly, by now, in the lowering significantly the morbidity and mortality of colorectal cancer the disease, in the world, and, after, with other organs and systems. RIBOGRAMA is an objective and quantitative method that provides a unique service to HealthCare Providers worldwide. The method is qualified to deal within the HealthCare worldwide, from the sole physician practice to the largest chain of hospitals. RIBOGRAMA is well suited to be addressed and recommended to many of the challenges facing HealthCare Providers worldwide in today's ever-changing business environments, to biomedical activity, life sciences and biosciences, and also offers a certain utility basis commonly in a cross section of "bio-industries" worldwide. The timing for this method to enter the domestic and global markets is understable. This method seems that will be a big solution for the people and the countries, through the world, to help the control (and part of the solution) of the cancer disease.

5 - MOTIVATION AND RATIONALE FOR THE METHOD 1. The CRC cancer is a leading cause of cancer death in the world with a Western lifestyle, but is within the best curable cancers when identified at an early stage; 2. This cancer has a long premalignant phase and a slow progression from the stage of disease confined to the wall of the organ to the stages of local invasion and distant metastatic disease.

Therefore, there is ample opportunity to identify patients at a curable stage with the completion of extensive tracking; 3. In a study tracking CRC explained the advantages and disadvantages of various methods of test or tests to 100 patients and then asked which method they preferred known as tracking. The results were that one third of patients decline any form of invasive test, but would undergo a noninvasive test; 83

RIBOGRAMA PROJECT 4. The CRC screening has proven effective both in reducing mortality from CRC, and, more recently, the decline in incidence.

5. There are certain conditions that diminish the potential of the results of the scans, which are associated with several factors, including: a) - The number of screening strategies; b) - Its varied patterns of recommendation for improper handling and inadequate stool blood test; c) - Excessive consumption of time and preparation uncomfortable for the patient in the early stages of bowel cleansing and d) - Invasive nature of many rigorous tracking procedures; 6. All these factors contribute to lower benefits and lower the overall value of the effort of inquiry; 7. CRC tracking has not penetrated adequately sampled population capable of being studied and indications for this, for example, older age 40/50; 8. The creation of clinical reminder systems for tracking CRC to ensure that patients remain faithful to tight tracking programs is equally necessary, and conditioned by the complexity involved in preparing the organization of crawls.

The method RIBOGRAMA allows to the patient, in a quantified way and non-invasive way, an easy adhesion of the population, by studying the pattern of the amount of free ribosomes in cells of colorectal mucosa, mixed with the stool. The harvest is made possible from a collector of feces, attachable to the toilet in the bathroom in the house of each patient that chooses to conduct the test, without the need to manipulate their own feces.

As is described in the Abstract, the method gives a concrete numerical result; is convincing to the patient's family and those with whom he speaks in the day-to-day, causing other people to be induced to do it, as with the control of diseases treatable and controllable.

The patient just take to his home the "collector" of feces (see the design of the colector in the final part of this proposal), which will buy in a pharmacy, in a chemist, or in a commercial area, and after the collection of faeces, guided by a small illustrated book of instructions, will deliver the device in a clinical laboratory, which should have technical suitability and accreditation. The practical consequences of this method are such that there is a marked decrease in health costs, resulting from the treatment of a very early colorectal cancer, because people know when to go to a medical appointment, when the RIBOGRAMA CURVE gives information, on early and useful time related their risk; may come to have the appearance of a malignant polyp, which arise as a result of a persistent increase in RIBOGRAMA CURVE before any colonoscopy.

Patients can repeat its RIBOGRAMA proof, within short intervals (without the risk of being surprised with a malignant lesion, or on the path to malignancy), at low cost, without having to be submitted to a blind colonoscopy, with its discomfort, its preparation, with enemas and cathartics, plus its costs.

Speaking of costs with the disease, once diagnosed in a stage of cancer that exceeded the limit of the submucosa, one can speak on multiple financial charges as a result of a colorectal cancer, which can enumerate below: 1.

2.

3.

4.

5.

6.

Charges related to the operating room; Charges for intensive care and/or recovery room for surgical patients; Global charges related to the patient's hospitalization; Charges related to chemotherapy and other drugs; Charges for follow-up examinations; The Social Security system no longer receives input from an active citizen and is payable to a disabled 84

RIBOGRAMA PROJECT 7. Usually the patient will not go alone to a consultation and treatment follow up, taking with him a family member or friend who will not work in his workplace, with loss of productivity To achieve the proof of concept of the RIBOGRAMA METHOD is essential to take into consideration which are the phases and/or techniques to deal with it, that is to say that is necessary to be created the conditions to support themselves the completion of the several phases and/or technologies listed in the following cast (from 01 to 11):

6 - EXECUTION AND STEPS TO ACCOMPLISH THE METHOD Turning to a more analytical way, about the structure of work to be done, for the counting of free ribosomes under study, soon one realizes that there are different levels/sections, which are crucial for the quantification of free ribosomes in a given unit of volume, to which we call STAGES to perform the method, will employ the following techniques and/or phases: 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

Harvest faeces (Collection of faeces) Collection of exfoliated colonocyts in a preservation serum Rupture of tissues and cells Homogenization Centrifugation Subcellular fractionation Differential Centrifugation Equilibrium sedimentation Preparation of free ribosomes Labeling of free ribosomes with fluorochromes Counting of free ribosomes with the use of flow cytometry with reverse-transcriptase qPCR or RT-qPCR.

Stage 01 â&#x20AC;&#x201C; (Collection of faeces) - for which was already designed an industrial prototype (see the design of the colector of faeces in the final part of this proposal), about which are drawings in 2D and 3D. Harvest faeces: It has been designed a collector to harvest the faeces sample, which is fitted into the opening rate of the toilet, which contains an easy closure device in opaque plastic-type material. Thus, the patient produces a dejection of feces, at home, directly to the appropriate container (adaptable to the closure rate and easy-zipper type), which is securely closed, without any manipulation of faeces for be sent to a laboratory that runs the techniques of the method.

This phase takes place in the patient's home.

Stage 02 - Collection of exfoliated colonocytes - The faeces are formed by a mixture of undigested alimentary waste, microflore, endogenous secretions and exfoliated cellular components of the intestinal tract.

The mucosal cells and tissue fragments are released in the feces in the intestinal lumen, and thus constitute the sample for the test. There are techniques of isolation and collection of viable exfoliated human colonocytes from the stools, which are extensively studied and standartized that with appropriate adaptations, are used for the preparation and production of free ribosomes.

Is the phase where cells are exfoliated from colorectal mucosal surface, covering the stool, dragged through the introduction of a manifold colonocytes in serum of preserving through an opening in a upper part of the superior cylinder, without handling of feces, resulting in a "washed feces". Given the "design" of how the collector was designed, it becomes easy to separate the "washed stool" of their own feces, through a system 85

RIBOGRAMA PROJECT of two cylindrical chambers connected by a unidirectional valve, which opens with a simple gesture according to the illustration of the instruction manual that comes with the kit for collecting stool. Thus, the "washed-feces" is drained to a lower cylinder to the collector of feces, which remain in the upper cylinder that is disposable. In the lower cylinder will be collected the washed stool with large amounts of colonocytes.

This phase takes place in the patient's home.

Stage 03 - Rupture of tissues and cells: subcellular fractionation process has two stages: - Rupture of cells or tissues, to obtain a cell lysate (or tissue) in which the desired fraction is in proper condition for purification.

- Separation of the desired fraction from the remaining components of the cell or tissue, using any criteria such as a density difference (gradient centrifugation or differential centrifugation), the presence of an antigen, etc.

- Techniques of tissue and cell breaking: The first step in the purification of most proteins and subcellular structures is the disruption of tissues or cells to obtain a cell lysate in which the protein or multiprotein complex subcellular structure is in conditions which permitted isolation. This is accomplished using procedures commonly known as soft mechanical homogenization. These methods produce the rupture of cell membranes, gently, thereby releasing the cellular contents. The most common homogenization procedures are: Sonication: Involves the application of ultrasound to a cell suspension. The intense agitation destroys the cell membranes. Depending on frequency, intensity and energy applied can also destroy and even subcellular structures solubilized protein complexes. Cold is usually applied to prevent overheating of the samples that could cause denaturation of proteins. Use of detergents for solubilisation cell membranes: Cells are passed through small diameter holes that cause the breakdown of cell membranes.

This phase takes place in the laboratory.

Stages 04 to 09 â&#x20AC;&#x201C; with use of standardized technology, existing for decades in routine laboratory techniques with biology.

Homogenizers: It comes to breaking the cells with the help of a rotary piston that fits perfectly with the thick walls of a special glass tube, the homogenizer. There are devices in which homogenization is calibrated separation between the plunger and the glass wall to produce homogenized with a particle size.

Subcellular fractionation: For subcellular fractionation, which can encompass a set of methods and techniques, aims to obtain pure fractions or enriched in a specific cellular component, be it an organelle (mitochondria, nuclei, peroxisomes, ribosomes ...), a fraction of membranes (total membrane, plasma basolateral domain, apical domain,...), or multiprotein complexes (actin cytoskeleton, microtubules, nuclear pores, etc. ..). The subcellular fractionation is a technique that involves the separation of different cell components by differential centrifugation. To do this, the cells are crushed and centrifuged several times. Each centrifuge separates an organelle distinct from the cell (nucleus 1). Observe the speed at which sediments may know each organelle sedimentation coefficient expressed in units of S (Svedberg) (e.g. 70s ribosomes). The cell fractionation is used to investigate the biochemistry and physiology of organelles outside the complex environment of the intact cell.

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Centrifugation: The centrifuges are tools to bring to the samples that produce strong forces shortly sedimentation of the particles have a density greater than that of their surrounding medium. In general differ depending on the margins of acceleration to the samples submitted to: - centrifugal (from a few g to approx 3000xg), SUPER (or high-speed centrifuges range from 2,000xg to 20,000xg) and ultracentrifuges (from 15,000xg to 60,0000xg).

The centrifuges are often control the chamber temperature to prevent overheating of the samples due to friction. In ultracentrifuges, extreme speed (over 100,000 rpm), makes it necessary to make a strong vacuum in the centrifuge chamber to prevent heating of rotor and sample. In a centrifuge determinant element 5 is the rotor spinning device and in which the tubes are placed.

The parameters to be present in any centrifuge, which determine the conditions, are: A. Volume of solution in centrifuge, which will determine the type of tubes and rotors to use.

B. Chemical nature of the solution, which will determine the nature of the tube to use C. Differential in density between the particle sedimentation and density of the medium in which it is located. In general, the higher the difference before (shorter and less force of acceleration) sediment.

When the difference is very small can be spun by hundreds of thousands of g for hours.

D. Centrifugation: Due to differences in size and density, each cell component is subject to a centrifugal force. The force generated by the centrifuge is expressed as relative centrifugal force (RCF) in units of g. The RCF is a function of centrifuge speed in revolutions per minute (rpm) and the particle distance from the axis of rotation.

Sedimentation rate: It is possible to exploit the difference in speed to sediment the particles for a spin in an environment where there is a density gradient, being lower in the top and largest at the bottom. After a while the different particle populations are at different depths 5 tube. Making a small hole in the bottom can collect different fractions containing the different populations separated. This is the foundation of the preparative ultra-centrifugation, which determines the settling velocity of a particle (measured in units Svedberg, S). Once the cells lysate or homogenate this proceeds to their subdivision. One of the most common techniques is centrifugation. It is based on spinning the tube at high speed so that the accumulation occurs in the bottom of the particles that tend to sink to have a higher density than the medium in which they find themselves. So, after centrifuging the sample, uniform, will have separated into two fractions: supernatant (homogeneous fraction that has not settled) and the pellet (which has been affixed to the bottom of the tube).

Differential Centrifugation: In differential centrifugation, the homogenate is centrifuged repeatedly at high speed, gradually sedimenting small particles. The first is a 600g centrifugation for 10 minutes, which settles the nuclear fraction. This pellet or ball containing the nucleus, intact cells and tissue. The next separation is the post-nuclear supernatant or liquid particles suspended in the pellet nuclear, which is transferred to another centrifuge tube and centrifuged at 10,000g for 30 minutes in a refrigerated centrifuge. The sedimented material called mitochondrial fraction contains mitochondria and micro bodies. Differential centrifugation is based on the existence of different particles in suspension that differ in density of the medium. If centrifuged in mild conditions (recently, little acceleration force) sediment particles larger and/or more dense. When the supernatant of the first centrifugation is centrifuged again in terms of more time and acceleration force, settles back denser particles present and so on. It may apply increasing severity in the centrifuge and get a collection of sediments that are successively particle fractions of different size and/or density.

Equilibrium Sedimentation: 87

RIBOGRAMA PROJECT A system is usually performed using the ultracentrifuge. Is the separation of particles according to their buoyant density. The sample above is available or mixed with a density gradient steeper than the previous case, which contains a very high concentration of sucrose or cesium chloride. On centrifugation each subcellular component will shift up or down until it reaches a position where its density equals that of their environment (position of neutral buoyancy) and did not move more. As a consequence, produce a series of discrete bands, the closest bottom of the tube contain larger particles with buoyant density. This method is also called equilibrium density gradient. Different substances are used to achieve the density gradients, such as sucrose, coffee, and cesium chloride. The gradients can be self-generated such as those based on cesium chloride formed in the centrifugation process itself, or preformed such as sucrose or percolator. In the latter case the preparation is done before centrifugation through a gradient-forming device that consists of two cells connected by the base and with agitation in solutions that fit with the two extremes of concentration. As is pulled from one another replaces the fluid removed and the concentration changes linearly. An alternative is discontinuous gradient centrifugation, which creates a stepwise gradient inside a tube to successively stacked volumes homogeneous solutions of different densities. In the interfaces of the different layers will accumulate floating particles (less dense) on the bottom but that sink (denser) in the upper layer.

This phase takes place in the laboratory.

Stage 10 - Consisting of covalent bonding of two particles - a ribosome and one molecule of a fluorochrome (labeling of ribosomes).

It should be noted that the translation of genetic information into polypeptides by the ribosome is a universal and fundamental cellular process. The structural and biochemical evidence of ribosome dynamics strongly supports its functional importance during protein synthesis. Are described conventional molecular biology techniques that allow separating cellular ribosomes (free and bound to membranes) from other subcellular organelles. There are several studies that describe various ways of labeling ribosomes. The preparation of ribosomes, depending on the tissue under study, can be done through some adaptive modifications, published in previous studies.

This phase (nanotechnology) takes place in the laboratory.

Stage 11 â&#x20AC;&#x201C; Counting of labeled free ribosomes, using flow sensing devices, which allows the count from 50 to 70 thousand particles per second.

This phase (flow cytometry) takes place in the laboratory.

This protocol actions must take place without omission of any stage, because the methodology of RIBOGRAMA is to be used in crop conditions that fit into a scenario that goes from the proximity of the patient to the lab to the living away from a collection center of the biological product (cells of the colonic mucosa in serum of preservation, which is to allow no autolysis of the same, achieving thus the integrity of the free ribosomes for the count). With graphical recording of results in successive readings will allow reading to check what the trend of proliferative cells in colorectal mucosa.

RIBOGRAMA is well suited to be addressed and recommended to many of the challenges facing HealthCare Providers worldwide in today's ever-changing business environments and biomedical (public health, medical and hospital practice, clinical and laboratory research, food industry, veterinary, oncologic research, pharmaceutical industry research and clinical trials etc), and also offers a certain utility basis commonly in a cross section of "bio-industries" worldwide. In summary, the timing for this method to enter the domestic and global markets is understable. This method seems that will be a big solution for the people and the countries, through the world, to help the control (and part of the solution of) the cancer disease.

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RIBOGRAMA PROJECT 7 - QUANTITATIVE PCR: TECHNICAL ADDENDUM Nowadays, quantitative polymerase chain reaction (qPCR) represents the method of choice for analyzing gene expression of a moderate number of genes in anywhere from a small number to thousands of samples. It is a robust and very well established technique based on the original design of the technique by Kary Mullis in 1983.

Standard PCR designed by Kary Mullis, is a technique that allows the selective amplification of DNA fragments. It involves the utilization of a thermo-stable DNA polymerase; a pair of oligonucleotide DNA primers that will hybridize and flank the amplified region; a template DNA molecule that contains the DNA segment to be amplified; a suitable buffer; and free desoxi-nucleotides. A typical PCR reaction is performed in three steps that will be subsequently cycled 30 to 40 times: Step 1 – DNA denaturation: the reaction mixture is heated up to 95ºC to completely denature all the DNA molecules, typically during 30-45 seconds.

Step 2 – Annealing: the reaction mixture is cooled down to 55-65ºC to permit the hybridization of the primers with the DNA template, also typically during 30-60 seconds.

Step 3 – Extension: reactions are heated again up to 70-72ºC, which is the optimum temperature of the polymerase, to perform the DNA extension. Time duration of this step will be dependent on the length of the fragment to be amplified.

Cycling these three steps a number of “n” times will generate a total of 2 n DNA molecules of the fragment to be amplified.

Classical PCR takes advantage of the thermal stability of the DNA polymerase that allows it to resist temperatures close to the boiling point of water. A typical PCR reaction is performed by the use of a specific piece of equipment called “thermocycler”. Modern thermocyclers are devices based in the peltier technology which permits very fast and accurate changes in the temperature of the samples.

Adaptation of standard PCR for quantitative purposes was only possible in the late 90’s. Quantitative PCR or real-time PCR (qPCR) is based on the same reaction mixture than the original standard protocol, however supplemented with an intercalating fluorescence compound that will bind to the double-stranded DNA during amplification. These intercalating agents are routinely used for DNA staining in agarose electrophoresis, and only emit fluorescence when they are inserted between DNA chains. qPCR systems read fluorescence during the PCR reaction in each sample, displaying a profile similar to that represented in Figure 1. At the beginning of the reaction, the number of DNA molecules increased slowly reaching after that a phase of exponential growth. At the end of the exponential phase, the number of DNA molecules amplified from the template reaches a plateau, consequence of the limitation of reagents. This plateau corresponds to the classical PCR, in which the main objective is to specifically amplify a DNA fragment. In Figure 1, the three represented curves of amplification reached the same plateau at the end of the PCR cycles, but they are all different considering the cycle in which they represent an exponential amplification. Following this approach in each curve we can define “Ct” as the PCR cycle in which a particular sample reaches this exponential phase of amplification.

Using the same conditions for the PCR reaction, the Ct value depends only on the initial amount of template in each sample, and is inversely correlated with this amount. This fact could be used to quantify the amount of DNA present in each sample, by relating Ct values with a standard reference of already known DNA concentration.

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Figure 1: representative profile of fluorescence intensity during a qPCR reaction. Sigmoidal curves represent the fluorescence in response to the concentration growth of the amplified DNA molecules during the PCR cycles. In each curve is possible to define a Ct as the PCR cycle in which the growth of the number of molecules is exponential. The absolute value of Ct is inversely related with the number of template molecules in the samples. Having an already known concentration of a reference DNA molecule, it is possible to construct calibration curves to relate Ct values with the template concentration.

DNA could be quantified by qPCR; however this method is more useful for quantify RNA levels, and consequently gene expression. For this purpose a previous enzymatic reaction is required in which RNA must be converted into a cDNA (complementary DNA) by the action of a reverse-transcriptase. This technique is often called reverse-transcriptase qPCR or RT-qPCR.

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XIX - CAPSULE I am available to do a lecture about the technico-scientific concept of the RIBOGRAMA PROJECT, to researchers, teachers, students and business men and investors on the topic RIBOGRAMA, focusing on various aspects related with the phenotypic behavior of a cell in a malignant process, to reveal the potential of the method, which will become in an Universal tool for research in oncology and other areas such as the toxicity of substances on the cellular homeostasy, in the pharmaceutical industry, in food industry, in agriculture and in ecologic systems.

With these thoughts on the method RIBOGRAMA, I intend to implement a project that would cover their study in clinical practice as applied to cancer of various organs in separate independent studies: colon and rectum, digestive tube, pancreas, breast, lung, urinary tract, prostate, endometrium, cervix and blood. It means that we are faced with a transversal new method of biomolecular diagnosis or harm to the human health, concerning the environment, as long as the human being lives in it, with it.

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XX - NUEVOS CAMINOS EN LA INVESTIGACIÓN DEL CANCER RIBOGRAMA: - Un nuevo concepto en la investigación biomolecular y su aplicación en la clínica oncológica y laboratorial del colon y recto.

El trabajo de investigación, que ahora se presenta es parte del capítulo xv del libro enunciado, ha sido elaborado en el ámbito de una Tesis Doctoral, en la Facultad de Medicina de la Universidad Complutense de Madrid, bajo la dirección del Profesor J.L.Balibrea Cantero, y tiene como objetivo el desarrollo del concepto RIBOGRAMA. Partiendo de la elaboración fundamentada, con técnicas y conceptos actuales de Biología Molecular, se llegó al concepto de RIBOGRAMA, que será un método de diagnóstico, rastreo y seguimiento de la enfermedad oncológica del colon y recto. Este método permite el diagnóstico “pre-carcinoma in situ” dado que se basa en el fenotipo de las células de la mucosa constituido por cantidades muy elevadas de ribosomas libres en su citoplasma, que se pueden cuantificar por medio de una técnica de conteo de partículas, con el mismo principio de la Citometria de Flujo, después de su aislamiento y marcados con fluorocromos. Los registros sucesivos de esas cantidades de ribosomas libres constituyen una curva gráfica que representa la tendencia de malignización (RIBOGRAMA), que por encima de un cierto nivel de concentración permite afirmar que las células de un tejido (del colon y recto p. ej.) se pueden considerar que están en un proceso de desarrollo de carcinoma en la mucosa colo-rectal, antes de cualquier visualización directa macroscópica (endoscopia). O sea, se está ante un método nuevo, a nivel biomolecular, evidente, como se demuestra en dicha Tesis, que permite una buena antelación sobre la macroscopia. Este método sobrepasa el ámbito de otros métodos no invasivos del colon-recto (test de sangre oculta en las heces y test de las mutaciones del ADN en las mismas heces, el último de los cuales comenzó a practicarse clínicamente hace poco más de cinco años en los EE. UU. de América y en Europa). Merced de este método es posible un programa de rastreo (por método no invasivo, con gran adhesión de la población), por el cual todas las personas con elevación significativa de la curva de RIBOGRAMA deben ser chequeados obligatoriamente con una endoscopia diagnóstico - terapéutica y al mismo tiempo permanecer bajo observación y terapia (dieta post-encuesta alimentaria y antiinflamatorios no esteroides y ácido acetilsalicílico, con demostrada eficacia en la prevención y disminución de la tendencia a la formación de pólipos o lesiones premalignas), con lo que ello representa de ahorro en la inversión terapéutica del cáncer.

El presente estudio parte de la observación de varias líneas de evidencia, algunas bien establecidas y recientes, que sostienen acentuadamente la idea de que el contenido de ARN esta elevado en las células cancerosas y que los eventos genéticos que llevan al cáncer están frecuentemente ligados, directa o indirectamente, a la biogénesis de los ribosomas (1,2). Por otro lado, el problema inverso se verifica en los casos de “anemia ineficaz idiopática” (IIE – idiopathic ineffective erythropoiesis), en que existen tasas insuficientes de proliferación celular, y los pacientes tienen eritroblastos que contienen solo 70% de los niveles normales de rARN (3). Investigadores han demostrado que las células cancerosas con expresión elevada de las proteínas ribosómicas tienen contenidos más elevados de ribosomas. Las alteraciones genéticas asociadas con el desarrollo del cáncer implican, muy frecuentemente, cambios de las vías de señalización que llevan su efecto hasta el rADN (4,5). El panorama anteriormente descrito reitera lo conocido por los biólogos del cáncer: la biogénesis de los ribosomas y la oncogénesis están íntimamente ligadas.

Nuestro propósito, con estos conocimientos básicos de biología molecular, es definir algunos principios orientadores sobre el valor del número aumentado de ribosomas libres, como traducción clínica de la dinámica del proceso bioquímico hacia la malignización de las células en una comunidad celular específica de un tejido, u órgano, mediante la verificación y comprobación en el ámbito de observaciones de microscopia electrónica de las cantidades de ribosomas libres en tejidos neoplásicos, o pre-malignos y de la construcción 92

RIBOGRAMA PROJECT de una curva grafica de las cantidades de ribosomas libres de esos tejidos (RIBOGRAMA), que pueda definir la tendencia de malignización de una comunidad celular, bajo ciertas condiciones que predisponen para el crecimiento de un tejido en la dirección de la malignización.

MATERIAL Y METODOS Realizamos un estudio retrospectivo de 206 casos de observación de microscopia electrónica en células y/o tejidos en estado de “premalignización”, o con fenotipo de malignidad, recopilados de la base de dados PubMed, referentes a descripciones de casos de diagnósticos anatomopatológicos bien establecidos y publicados, en revistas idóneas y creíbles de la comunidad científica que abordan temas de biología molecular, oncología, y temas de las ciencias biomédicas, existentes en la literatura científica de las ciencias biológicas y de la salud. Esas descripciones tienen referencia a células y/o tejidos tumorales malignos (o con posibilidades de seren comparados con tejidos de crecimiento maligno). De las descripciones de eses casos seleccionados fueron recopilados los principales datos descriptivos de la morfología encontrada en la respectiva observación de microscopia electrónica, especialmente en los elementos morfológicos de la textura de la célula, como por ejemplo, los organelos celulares.

En su globalidad cada célula de un clone maligno ofrece una buena idea sobre la morfología de un fenotipo celular maligno. La organización y registro de los datos descriptivos de las observaciones de microscopia electrónica, coleccionados de la base de dados PubMed, constan de la inscripción en la cual se trazaron cinco columnas, correspondientes a los siguientes cinco ítems (TABLA I – ANEXOS): A. Número de orden del caso bajo estudio; B. Descripción cuantitativa de los ribosomas libres de las células de un determinado tejido, o órgano bajo estudio; C. Aspectos genéricos resumidos de la descripción, en términos de observación en microscopia electrónica, de los principales organelos celulares; D. Información del diagnóstico anatomopatológico del tejido, u órgano, bajo estudio, E. Referencia a la bibliografía constante en la literatura médica de referencia científica (creíble y aceptada por los medios de investigación científica y / o instituciones de ID).

OBJETIVO Descripción de los aspectos cuantitativos relativos a los ribosomas libres de las células de un determinado tejido, o órgano, bajo estudio: En la columna del ítem “descripción cuantitativa de los ribosomas libres” de las células de un determinado tejido u órgano se encontraron algunos grupos de expresiones, en un universo de 206 observaciones, que han sido utilizadas repetidamente por los investigadores de microscopia electrónica, que esencialmente se distribuyen por seis grupos:

I - NUMEROUS FREE RIBOSOMES ...........................................................................36 casos II - RICH IN FREE RIBOSOMES .................................................................................. 05 casos III - ABUNDANT FREE RIBOSOMES .......................................................................... 36 casos IV - MANY FREE RIBOSOMES .................................................................................... 19 casos V - INCREASED NUMBER OF FREE RIBOSOMES ................................................. 28 casos VI - FREE RIBOSOMES ................................................................................................ 70 casos 93

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Las expresiones “numerous free ribosomes”, “rich in free ribosomes”, “abundant free ribosomes”, “many free ribosomes” y “increased number of free ribosomes”, utilizadas en las descripciones de microscopia electrónica de la TABLA 1 - ANEXOS son equivalentes, pero son subjectivas, porque no permiten una reproducibilidad numérica/matemática de resultados. Algunas otras expresiones han sido utilizadas en la TABLA 1 – ANEXOS, que por no seren tan frecuentes en el conjunto de los 206 casos coleccionados, no han sido destacadas.

En el grupo VI, con 70 casos (correspondiente a 33,7% del total de los 206 casos seleccionados, con descripción morfológica en microscopia electrónica), en que es utilizada la expresión “free ribosomes”, no hay una idea de la cantidad (aumentada o disminuida) de los ribosomas libres en las descripciones de microscopia electrónica, a pesar de los diagnósticos anatomopatológicos descritos en el cuadro VI de ANEXOS. A través de los otros 124 casos (correspondientes a 59,6% del total de los 208 casos seleccionados con descripción morfológica en microscopia electrónica) en que son utilizadas las expresiones “numerous free ribosomes”, “rich in free ribosomes”, “abundant free ribosomes”, “many free ribosomes” y “increased number of free ribosomes” es posible tener una idea de que hay un aumento significativo de la cantidad de ribosomas libres, a pesar de subjetiva, y hacer una apreciación de que en los diagnósticos de malignidad correspondiente a esos casos, como consta de los cuadros I, II, III, IV, V ( en los ANEXOS) hay una constancia de la característica constituida por un aumento de ribosomas libres en las células que están en un proceso de multiplicación sin control de autorregulación, como en el caso de las células malignas. En la TABLA 1 – ANEXOS está la descripción de microscopia electrónica de los organelos de células obtenidas a través de una busca en la base de dados PubMed, mediante la utilización de parámetros de busca asientes en las expresiones “free ribosomas” y “cancer cells” En cada uno de los ”grupos de expresiones” se hizo el conteo del número de casos de tejidos o órganos con fenotipo de malignidad confirmada y inequívoca, relativamente al total de la muestra de ese grupo. Se obtuvo, así, el porcentaje de tumores y/o tejidos malignos para cada uno de los seis “grupos de expresiones”, como consta en la TABLA II:

Cuadro de los “grupos de expresiones” I

TABLA II Muestra de cada Fenotipos de malignidad correspondientes a cada “grupo de “grupo de expresiones” expresiones” 36

33

% de tejidos o tumores malignos 92 %

II

5

5

100 %

III

36

34

94,4 %

IV

19

16

84,2 %

V VI

28 70

22 59

78,6 % 84,29 %

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RIBOGRAMA PROJECT Con los resultados presentados en la Tabla II se verifica que la cantidad/densidad de ribosomas libres constituye uno de los aspectos fenotípicos fundamentales y evidentes de una célula con características de malignidad. Entonces, lo importante será buscar un lenguaje no subjetivo, reproducible, con traducción matemática, a través de una curva gráfica a que se llamará RIBOGRAMA. Tal curva traduce una idea dinámica de las tendencias de las variaciones de los valores o cantidades numéricas del conjunto de los ribosomas libres. La cantidad/densidad de ribossomas libres constituye el aspecto fenotípico de la tendencia dentro de la normalidad, o hacia la malignidad, de una célula o comunidad de células bajo observación en lo que respecta a su comportamiento biomolecular, lo que representa un fenotipo mutante con crecimiento maligno.

DISCUSIÓN En las décadas de 60 y 70 fueron publicados estudios sobre ribosomas libres y ribosomas ligados a membranas (22,23) y sobre su acumulación durante la inducción de crecimiento en muchos órganos y tejidos (17-20). En la secuencia de estas investigaciones se publicaron estudios que han intentado cuantificar la acumulación de ribosomas en las zonas interfoliculares de la piel del dorso de ratones durante la inducción química de crecimiento neoplásico, en dos fases, mediante iniciación por 7,12-dimetilbenz(a)antraceno y promoción provocada por 12-O-tetradecanoil-forbol-13-acetato (25).

La epidermis es un epitelium de superficie y, como otros epitelios, que están en contacto directo con el medio externo, tales como los revestimientos de los aparatos respiratorio y gastrointestinal, muestra una elevada incidencia de neoplasias (21). De este modo, la epidermis ha servido como un modelo útil para constituir un sistema de desarrollo comprensible del papel de la acumulación de ribosomas durante el crecimiento neoplásico (24).

Será muy importante la producción de modelos predictivos mecanísticos ( conteo de ribosomas libres) basados en la dinámica tumoral, con traducción en el fenotipo celular, como, por ejemplo, con el conteo de ribosomas libres en una célula en proceso de alteración fenotípica, bajo el estímulo oncogénico. Será más útil para la práctica clínica concreta que los investigadores puedan abstraerse del significado de los detalles biológicos más avanzados relativamente a la oncogénesis y progresión tumoral, que dependen de incontables factores variables. Como muchos campos de las ciencias de la vida, la biología del cáncer es un campo en expansión exponencial y complejo, envolviendo trabajo que tiene una amplitud desde la biología molecular de los oncogenes hasta la epidemiología del ambiente. Las tasas de supervivencia para los varios canceres, una vez que se manifiesten clínicamente, han mostrado modesta mejoría durante las últimas décadas.

La identificación de los cambios de la estructura del citoplasma, como por ejemplo el aumento acentuado del número de ribosomas (método cuantitativo) acumulados en el citoplasma de una comunidad de células de un determinado tejido puede constituir uno de los aspectos fenotípicos de la expresión de malignidad de la estructura fina de las células transformadas, lo que puede ser esencial para caracterizar la evolución del comportamiento celular. En este sentido, por ejemplo, se podría estudiar el perfil gráfico de los cambios del número de ribosomas por célula (o por unidad de volumen), en un período de tiempo definido, dentro de una media aritmética de las células exfoliadas de la mucosa colorrectal de un paciente cuyos registros secuenciales formarán una curva patrón/media del número de ribosomas por unidad de volumen (RIBOGRAMA), con relación a las células de la mucosa del colon y recto, aisladas y separadas de las heces.

Esto puede permitir una fuerte motivación para integrar diversos campos del conocimiento en la biología del cáncer e introducir nueva armazón conceptual y teorética que pueda mejorar la comprensión de los investigadores sobre la dinámica de la formación tumoral para, así, poder desarrollar mejores métodos preventivos, diagnósticos y terapéuticos.

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RIBOGRAMA PROJECT Aunque sea cierto que el cáncer es una enfermedad multifacetada con una variedad de “triggers” cercanos en diferentes tejidos y en diferentes pacientes, hay también una fuerte posibilidad que los cánceres comparten una funcionalidad central originándose a partir de una maquinaria celular común de la cual las células dependen para su proliferación (11). El aspecto más visible de los neoplasmas malignos agresivos es la proliferación celular aumentada, la cual tiene en su base un acentuado aumento de la síntesis proteica. En los procesos de la respuesta mitogénica normal hay un aumento transitorio y cíclico del índice de la síntesis genérica de proteínas. El aumento general en la síntesis proteica es un fenómeno necesario controlado que se observa antes de la división celular, llevando a la duplicación del contenido y al aumento del tamaño antes de la mitosis normal. Así, el tamaño medio de las células es mantenido durante el proceso de respuesta proliferativa fisiológica. Uno de los mecanismos llave de la pérdida del control de la síntesis proteica en las células transformadas es la incapacidad de disminución del número de ribosomas que esta correlacionada con la proliferación celular en medios de cultura frescos sin adición de factores de crecimiento al suero (6).

Muchos investigadores han observado, en culturas de tejidos, diferencias en las propiedades de crecimiento entre células normales y sus contrapartes malignas, una de las cuales apunta hacia el fallo de estas en mostrar una variación cíclica de algunos parámetros celulares y bioquímicos a través del ciclo celular o ciclo de crecimiento (7-10).

Anteriormente, otros investigadores han prestado su atención a las modificaciones que ocurren en el ritmo de la síntesis proteica y en la función de la maquinaria “translacional” de la célula, relativamente al ciclo celular, una vez que tales cambios son necesarios en las transiciones de crecimiento y multiplicación en condiciones normales. En tumores de la piel en ratones, inducidos por la aplicación de promotores de tumores, la relación ARN: ADN y el contenido de ARN (porcentaje de masa seca contribuida por ARN) eran 2 a 3 veces más elevadas que en los tejidos normales (12). En varios tipos de leucemia, el contenido de ARN de linfocitos estaba fuertemente correlacionado con la cinética del crecimiento celular acelerado y con el pronóstico del enfermo (13).

En un estudio de cánceres ginecológicos en que los tejidos neoplásicos fueron comparados con sus contrapartes normales, el contenido de ADN y el contenido de ARN en los tejidos neoplásicos estaban aumentados 1.6 y 2.4 veces, respectivamente (14). De igual modo, el contenido de ARN celular estaba aumentado por un factor de 1.4 en células de neuroblastoma myc-transfectadas relativamente a las células normales (15). En otro estudio de cáncer de mama el test del contenido de ADN celular estaba normal, pero el contenido de ARN celular estaba bien correlacionado con el grado del tumor, con el tipo histológico, con el status hormonal y con la supervivencia del paciente (16).

Biogénesis de los ribosomas La biogénesis de los ribosomas y el control de la traducción son procesos celulares esenciales controlados a muchos niveles. Varios supresores tumorales y proto-oncogenes han sido responsabilizados por la alteración de la formación de los ribosomas maduros y por la regulación de la actividad de proteínas conocidas como factores de traducción. La perturbación en una o más de las etapas que controlan la biosíntesis de las proteínas ha sido asociada con alteraciones en el ciclo celular y en la regulación del crecimiento de las células. Por eso, ciertos supresores tumorales y proto-oncogenes pueden regular la progresión maligna a través de la alteración de la maquinaria de la síntesis proteica.

La producción de ribosomas maduros, que son competentes para la traducción celular del mARN necesita de un proceso “multistep” que es altamente coordinado en las células eucariotas. El ribosoma, que es la fábrica central de síntesis de proteínas, puede ser visto como una máquina finamente regulada que funciona como un componente estático de los complejos procesos centrales ordenados a niveles más superiores. De hecho, los ribosomas tienen la misión de producir correcta y eficientemente todas las proteínas de la célula. Aunque 96

RIBOGRAMA PROJECT sea conocido el hecho de que en las células cancerosas los componentes de la maquinaria de traducción están desarreglados o se expresan mal, su papel en la tumorigénesis ha sido largamente olvidado. Por ejemplo, en los comienzos de los años 70, los cambios en el nucleolo han sido reconocidos como un importante marcador de la transformación celular (26).

Las mutaciones en los genes que codifican las proteínas que están directamente envueltas en la biogénesis de los ribosomas están asociadas con el cáncer y otras enfermedades. El gen DKC1 (Dyskeratosis Congénita) está mutado en pacientes con la dyskeratosis congénita, que es una enfermedad caracterizada por enviejez prematura y un aumento de la susceptibilidad para el cáncer (27,28). El DKC1 codifica la disquerina, una sintasa pseudouridina que hace la mediación post-transcriptional del ARN ribosómico. Se identificaron mutaciones en el gen que codifica la proteína ribosómica S19 en otro síndrome que es caracterizado por un aumento de la susceptibilidad para el cáncer – Anemia de Diamond-Blackfan (29).

El crecimiento y proliferación celular están asociados con cambios en la tasa de producción de los ribosomas.

Durante G1 hay un prerrequisito que es el aumento de la síntesis del rARN y del montaje de los ribosomas para el aumento de la síntesis proteica durante la fase S (30). Más aún, puede ser necesario la regulación de baja en la actividad de los ribosomas o de su formación, o ambos durante la fase M para asegurar la salida adecuada del ciclo celular (31,32). Por eso, existe una relación importante entre el ciclo celular y la producción de los ribosomas.

Este balance es mantenido en la célula a través de los “checkpoints”, que aseguran que la traducción del mARN ocurra en niveles apropiados y durante una ventana del ciclo celular. La síntesis del rARN es el primero evento en la biogénesis del ribosoma. Está dependiente de la regulación del rADN por la ARN polimerasa I (Pol I) en el nucleolus. La síntesis de rARN en la célula puede ser inducida por estímulos extracelulares en ciertos momentos cuando una célula necesita de crecer y proliferar.

El concepto de la regulación de la síntesis del rARN ha sido originalmente verificado en las células en que la privación de un aminoácido resultaba en una rápida terminación de la síntesis del rARN (33). Desde entonces, muchos otros artículos han mostrado que la iniciación de la transcripción de rARN está íntimamente ligada a la progresión del ciclo celular. La síntesis del rARN es máxima en las fases S y G2 y reprimida en la mitosis y aumentada en G1 (32, 34, 35).

Estas fluctuaciones en las síntesis de rARN dependientes del ciclo celular están dependientes de la actividad de Pol I. La trascripción del factor UBF (upstream binding factor) es una llave reguladora de la síntesis de rARN teniendo la capacidad de modular la actividad transcripcional de Pol I (36-38). Más aún, varios protooncogenes y supresores de tumores regulan directamente la síntesis de rARN por potenciación o represión de la actividad del UBF, respectivamente.

La primera proteína identificada que regula la actividad de UBF ha sido la CKII - casein kinase II (41), una quinase serina treonina que tiene aumentada su expresión en muchos cánceres, incluyendo leucemias y tumores sólidos. La CKII ha sido responsable de contribuir a la tumorigénesis a través de la interacción directa con la maquinaria del ciclo celular. Además, esta fosforila el UBF en el carboxilo terminal y por eso regula la trascripción del rADN. Hay estudios subsecuentes que han estudiado otras quinases de UBF que también están desarreglados en el cáncer y afectan similarmente la síntesis del rARN (39,40). Se sabe, desde hace más de 25 años, que la tasa de proliferación y crecimiento celular es proporcional a la tasa de síntesis proteica (42,43). Además, la desregulación aumentada de las quinases UBF en los cánceres puede estimular la síntesis de rARN y contribuir a sus propiedades oncogénicas. Por eso, la perturbación del control de la síntesis proteica puede tornar las células más susceptibles para desarreglar el crecimiento y proliferación celular.

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RIBOGRAMA PROJECT La desregulación aumentada de las r-proteínas en las células cancerosas corresponde razonablemente a su envolvimiento en la producción de ribosomas. Similarmente, al aumento de la actividad transcripcional de Pol I que resulta en un aumento de la síntesis de rARN, las r-proteínas podrían también reglar el número de ribosomas funcionales en la célula. En ambos casos las células que contienen más ribosomas tendrían una tasa de traducción aumentada, que promovería la transformación celular (44,45).

El crecimiento y proliferación celular están asociados con cambios en la tasa de producción de los ribosomas y la biogénesis de los ribosomas y puede servir como un sensor para que las células ultrapasen importantes “checkpoints” durante el ciclo celular. En las células transformadas, que muestran producción de ribosomas aumentada el ciclo celular, el número de ribosomas puede ser alterado como consecuencia de las alteraciones en la biogénesis de los ribosomas. Este sistema apretado de autorregulación entre los ribosomas y el ciclo celular podrá funcionar para mantener la homeostasis celular. Por eso, un aumento en la cantidad de ribosomas, como consecuencia de algún efecto a montante, afecta la traducción de las proteínas y puede contribuir para el proceso de transformación. Sin embargo, en el presente, no es posible saber en concreto se hay algún “cross-talk” entre los ribosomas y el ciclo celular.

Consideraciones teóricas sobre una curva gráfica de RIBOGRAMA En el esquema de la fig. 1.15 están diseñados los dos ejes que representan las dos coordenadas cartesianas (abscisa o eje horizontal y ordenada o eje vertical), relativamente a los cuales se va construir una curva gráfica. Esta curva es caracterizada por dos variables relativas a los ribosomas libres de las células de la comunidad citológica bajo estudio (las células epiteliales de la mucosa colorrectal o coloncitos). Esas variables son: a) - la “cantidad (número) media de ribosomas libres” en el citoplasma de cada célula y b) - el “tiempo” a lo largo del cual es observado el comportamiento de ciertas variaciones de esa “cantidad (número) media de ribosomas libres”.

Habrá necesidad de criar experimentalmente perfiles gráficos de valores numéricos de la “cantidad (número) media de ribosomas libres” que puedan ser fijados como normales, a partir de los cuales se pueda trazar una curva-mediana-padrón. Así, será posible hacer estudios comparativos de grandes grupos poblacionales, en los cuales será posible divisar una multiplicidad potencial de tendencias de enfermedad maligna (por elevación de la curva de ribograma) en una determinada comunidad de células de un tejido bajo investigación.

En estudios correlacionados de microscopia electrónica y bioquímica los datos bioquímicos son usualmente cuantitativos, mientras la información morfológica (de la microscopia electrónica) es más limitada o semicuantitativa, basada en descripciones dependientes de criterios subjetivos. Este criterio subjetivo de abordaje en la microscopia electrónica no permite una mensuración estadística de los datos, con rigor matemático, impidiendo una correlación paramétrica de los datos morfológicos con los correspondientes datos bioquímicos.

En el ribograma hay un registro de la evaluación cuantitativa de los ribosomas libres, en el sentido estático y dinámico, como información recogida de fracciones subcelulares, que puede ser correlacionada con otros parámetros de mensuración biológica celular cuantitativa (como por ejemplo, algunos marcadores tumorales, e.g. CEA.). A partir del conocimiento del intervalo de valores de la “cantidad (número) media de ribosomas libres” considerados dentro de la normalidad (intervalo A) se podrán considerar otros intervalos (niveles), que en la fig. 1.15 están representados por B, C y D. En el eje de las abscisas se inscriben los múltiples de períodos de seis meses (smt), contados a partir del inicio de la monitorización de los registros de los valores de la “cantidad (numero) media de ribosomas libres” de las células bajo estudio.

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RIBOGRAMA PROJECT Características de la malignidad versus conteo de ribossomas libres.

Por el contenido de la Tabla 1.15 se verifica que las descripciones de ME y del comportamiento de las células con características fenotípicas de malignidad varían según el grado de menor o mayor diferenciación de su textura, o morfología, pero esas descripciones son subjetivas, dependiendo de cada observador que hace la investigación. Con el transcurrir del tiempo, el número de ribosomas libres sufre una variación progresiva de la cantidad (disminución), cuando las células epiteliales se desplazan del fondo de una cripta intestinal hasta al vértice de la vellosidad (Fig. 2.15).

Fig. 2.15 En términos de biología molecular del cáncer, la transformación maligna de las células normales consiste en la adquisión progresiva de una serie de cambios genéticos específicos que actúan desobedeciendo a los fuertes mecanismos antitumorales que existen en todas las células normales, los cuales incluyen: a) regulación de la transducción de señales; b) - diferenciación celular; c) - apoptosis; d) - reparación del ADN; e) - progresión del ciclo celular; f) - angiogénesis; g) - adhesión celular, y no son cuantificables.

Al igual de las descripciones de ME, estos mecanismos de transformación maligna sólo son evaluados a través de criterios subjetivos, en que las características descritas no pueden ser cuantificables matemáticamente. De igual modo, las propiedades de las células transformadas malignas, en crecimiento, en cultura de células, o in vivo, cuando observadas según técnicas de biología celular, o de biología molecular, no posibilitan una evaluación cuantitativa rigurosa, porque dependen de criterios subjetivos, sin límites numéricos, como son las propiedades enumeradas en el Cuadro 1.15 (propiedades de las células transformadas malignas creciendo en cultura de células y/o in vivo). Con los ribosomas libres es posible cuantificarlos, a través de conteo, utilizando técnicas de citometria de flujo y comparando los resultados con la variable tiempo.

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CUADRO 1.15 - Propiedades de las células transformadas malignas creciendo en una cultura de células y/o in vivo 1. Alteraciones citológicas parecidas con las células cancerosas in vivo, incluyendo basofilia citoplasmática aumentada; número y tamaño de los núcleos aumentado; relación núcleo-citoplasmática aumentada; formación de clusters y cordones de células; 2. Alteración en las características de crecimiento: a. “Inmortalidad” de la células transformadas en cultura. Las células malignas transformadas se tornan “inmortales”, en que ellas pueden ser transferidas en cultura indefinidamente. b. Disminución de la inhibición dependiente de la densidad celular o pérdida de la “inhibición de contacto”. Las células transformadas frecuentemente crecen hasta una densidad más elevada que sus contrapartes normales, y ellas pueden amontonarse en cultura en vez de parar de crecer cuando entran en contacto entre ellas. c. Disminución de las necesidades de suero. Las células transformadas necesitan de concentraciones disminuidas de suero o de factores de crecimiento para replicarse en cultura comparativamente a las células no transformadas. d. Pérdida de la dependencia para anclar y adquisición de la capacidad para crecer en agar blando. Las células transformadas pueden perder su necesidad para crecer adheridas a las superficies y pueden crecer como colonias libres en medio semisólido. e. Pérdida del control del ciclo celular. Las células transformadas no paran en G1, o en el límite G1/S del ciclo celular cuando están sujetas a restricciones metabólicas de crecimiento. f. Resistencia a la apoptosis (muerte celular programada).

3. Cambios en la estructura y función de la membrana celular – incluyendo aglutinación aumentada por las lecitinas de las plantas, alteración en la composición de las glicoproteínas de la superficie celular, proteoglicanos, glicolípidos y mucinas; aparecimiento de antígenos asociados a tumores; y aumento de la captación de aminoácidos, hexoses y nucleótidos.

4. Pérdida de las interacciones célula-célula y célula-matriz extracelular que fomenta la diferenciación celular.

5. Pérdida de respuesta anti agentes inductores de diferenciación y receptores celulares alterados para estos agentes.

6. Alteración de los mecanismos de transducción de señal, incluyendo receptores de crecimiento constitutivos, cascadas de fosforilación y mecanismos de defosforilación en vez de una función regulada.

7. Expresión aumentada de proteínas oncogénicas debido a translocación, amplificación y mutación cromosómicas.

8. Pérdida de productos proteicos de genes supresores de tumores debido a delección o mutación.

9. Errores de lectura genómica que llevan a la sobreproducción de substancias promotoras de crecimiento, e.g., IGF-2.

10. Aumento o producción desreglada de factores de crecimiento, e.g., TGF-alfa, factores de angiogénesis tumoral, PDGF, factores de crecimiento hematopoiético (e.g., CSFs, interleucinas).

11. Inestabilidad genética, llevando a la pérdida progresiva de la proliferación celular reglada, invasión aumentada y potencial metastático aumentado. Los genes “mutadores” pueden estar envueltos en este efecto.

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RIBOGRAMA PROJECT 12. Alteración en los cuadros enzimáticos. Las células transformadas tienen niveles aumentados de enzimas envueltos en la síntesis de ácidos nucleicos y producen niveles más elevados de enzimas líticos, e.g., proteasas, colagenasas y glicosidasas.

13. Producción de productos génicos de oncodesarrollo. Muchas células transformadas malignas creciendo en cultura o in vivo producen cuantidades aumentadas de antígenos oncofetales (e.g. antígeno carcinoembriónico), hormonas placentales (e.g., gonadotropina coriónica), o isoenzimas de tipo feto-placental (e.g., fosfatasa alcalina placental).

14. Capacidad para producir tumores animales experimentales. Esta es la condición sine qua non que define la transformación maligna in vitro. Si las células que se piensa han sido transformadas no producieren tumores en animales huéspedes apropiados, entonces no pueden ser definidas como “malignas”. Sin embargo, el fallo para crecer en un modelo animal no excluye el hecho que ellas puedan ser tumorigénicas en un animal de tipo diferente.

15. Capacidad para evitar respuesta inmune antitumoral del huésped.

De las características que se acaban de enumerar, al evolucionaren en la variable tiempo, no es posible obtener un registro numérico, que pueda constituir un conjunto de valores a través de una expresión gráfica o tendencia, que traduzca matemática y reproduciblemente la tendencia dinámica del estado de crecimiento y proliferación de un conjunto de células pertenecientes a un dado tejido, lo que pasa a ser posible con el concepto RIBOGRAMA.

CONCLUSIONES 01- Existe evidencia bien establecida (y reciente) que sostiene acentuadamente la idea de que el contenido de ARN está elevado en las células cancerosas y que los eventos genéticos que llevan al cáncer están frecuentemente ligados, directa e indirectamente, a la biogénesis de los ribosomas; 02- Así, el aumento significativo del número de ribosomas libres acumulados en el citoplasma de una comunidad de células de cualquier tejido (mediante el conteo con un citómetro de flujo, después de aislados y marcados con partículas fluorescentes --- fluorocromos) puede constituir uno de los aspectos fenotípicos de la expresión de malignización de la estructura fina de las células transformadas; 03- En las células en proceso de malignización, el aumento del número de ribosomas libres arriba de un cierto nivel de concentración (por unidad de volumen), que sea establecido como límite del padrón de normalidad, constituí un signo cuantificable de la tendencia de la expresión de malignidad. La verificación de ese aumento se observa antes que la estructura morfológica fina global de una célula transformada, o del tejido al cual pertenece, asuma un carácter instalado de malignidad; 04- La expresión fenotípica del citoplasma, representada por el aumento significativo de ribosomas libres, puede ser una característica del crecimiento neoplásico y podrá representar un aspecto preditivo de la tasa de proliferación celular y de pronóstico de los pacientes; 05- En los mamíferos hay evidencia de que existen cuatro tipos principales de células diferenciadas en el epitelio del intestino delgado: columnar velloso, mucoso, entero-endocrino y células de Paneth, que se originan del mismo precursor, que es un componente de la familia denominada por células criptobase. Estas células precursoras tienen carácter proliferativo, que en la mucosa del colon descendiente están localizadas en la base de la cripta, mientras en el colon ascendiente se encuentran en medio de la cripta; 06- Estas células epiteliales precursoras del tubo digestivo pasan a través de varios estadios, empezando como “stem cells”, que se dividen y producen nuevas “stem cells” y células como las células precursoras “no-comprometidas” y/o “comprometidas”, y son las responsables por la autorenovación continua del epitelio gastrointestinal a lo largo de la vida; 101

RIBOGRAMA PROJECT 07- Las “stem cells” comprometidas, que son células epiteliales especializadas por su capacidad de automanutención, proporcionan la base de replazamiento celular en desarrollo. Estas células se dividen asimétricamente y producen una “stem cell” que se mantiene multipotente e indiferenciadas y una célula hija, que está comprometida con la diferenciación; 08- Las células hijas comprometidas sufren varios ciclos de división celular adicionales, mientras migran de un compartimiento proliferativo hacia un compartimiento diferenciado. Por eso, en el sistema gastrointestinal maduro hay, en todos los momentos, ya sea “stem cells” pluripotentes, que son células indiferenciadas, o células diferenciadas, que son células epiteliales funcionales. Más del 85% de todo el tejido epitelial en el colon comprende los coloncitos; 09- Estudios morfológicos y bioquímicos sugieren que los ribosomas de las células de los mamíferos están libres en la matriz citoplasmática (ribosomas libres) o ligados a las membranas citoplasmáticas que forman parte del reticulum endoplasmatico rugoso (ribosomas ligados a membranas); 10- Hay evidencia morfológica y fisicoquímica de que los tejidos bien diferenciados difieren de los pobremente diferenciados en que los últimos tienen una proporción más elevada de ribosomas libres; 11- La producción de los ribosomas es una actividad metabólica importante ligada al crecimiento celular y recientes datos sugieren que el nucleolo también juega un importante papel en la regulación del ciclo celular, en la senescencia y en las respuestas al stress; 12- La biogénesis de los ribosomas envuelve la síntesis de rARN, la maduración y el “assembly” del ARN y de las proteínas ribosómicas en las sub-unidades ribosómicas, pequeña y grande; 13- La biogénesis de los ribosomas es regulada a través del ciclo celular, primariamente al nivel de la síntesis de rARN; 14- Están desarrolladas investigaciones por oncobiólogos que han demostrado que la expresión de las alteraciones del rADN y de los genes de las proteínas ribosómicas están asociadas con el desarrollo de tumores, así como varios estudios citológicos evidencian que los nucléolos (los sitios en donde el rARN es sintetizado y los ribosomas son “assembled”) de las células cancerosas están aumentados porque tienen una actividad transcripcional aumentada, representando aspectos predictivos de la tasa de proliferación celular y del pronóstico de los pacientes, motivo por el cual, las células cancerosas tienen una elevada expresión de las proteínas ribosómicas, revelando contenidos más elevados de ribosomas, lo que explica que la biogénesis de los ribosomas y la oncogénesis están íntimamente conectadas; 15- Por su dimensión (25 nanómetros) el conteo de los ribosomas libres sólo puede ser estudiado con el recursos a la nanotecnología; 16- Con el presente estudio se demuestra que es posible definir algunos principios orientadores sobre el valor y el significado del aumento del número de ribosomas libres arriba de un cierto nivel de concentración, establecido como el límite de la normalidad; 17- Este aumento significativo progresivo del número de ribosomas libres representa la traducción clínicolaboratorial de la dinámica del proceso bioquímico hacia la malignización de las células en una comunidad celular específica de tejido, u órgano; 18- La verificación en el ámbito de observaciones de microscopia electrónica muestra la existencia de grandes cantidades de ribosomas libres en tejidos neoplásicos o pre-malignos. Con la citometria de flujo también se comprobará lo mismo, pero de modo cuantificable, después del aislamiento, marcación con colorantes fluorescentes y cuantificación de los ribosomas libres, lo que permite comparaciones de valores; 19- La comprobación de esas grandes cantidades de ribosomas libres puede ser traducida en una curva gráfica elevada que representa y define, a lo largo del tiempo, la predisposición para la malignización del crecimiento de un tejido, o comunidad celular, bajo ciertas condiciones; 20- Esa curva gráfica pasará a tener la designación de RIBOGRAMA. Tiene una base matemática (pudiendo ser reproducible en circunstancias equivalentes) que corresponde a un lenguaje no subjetivo; 102

RIBOGRAMA PROJECT 21- El análisis de las tendencias y variaciones encontradas en esa curva elevada constituirá un indicador seguro sobre el grado de evolución y propensión para la malignización; 22- El RIBOGRAMA corresponde a un aspecto fenotípico, que puede proporcionar una información cuantitativa, por niveles de riesgo y de significación, con antelación a la malignización “abierta”, y con información sobre el perfil de riesgo heredofamiliar y del perfil alimentario, dietético y de hábitos de vida, en cualquier tejido a estudiar cuanto a su dinámica proliferativa.

23- Comparativamente a los tests no invasivos existentes en uso (análisis de sangre oculta en las heces y análisis de mutaciones de ADN de los coloncitos) el test del conteo de ribosomas tiene características que constituyen grandes ventajas sobre los tests existentes: i.

Sabiendo que se van a estudiar coloncitos aislados según una técnica específica, la especificidad de los resultados es de 100% relativamente al conteo de los ribosomas libres; ii.

La existencia de niveles de cantidades de ribosomas libres, según criterios de riesgo y significación, permite antelación de actitudes preventivas y terapéuticas ante una tendencia de malignización, mientras que en el test de descubrimiento de mutaciones del ADN en las heces la respuesta es solamente positivo/negativo para determinadas mutaciones, no permitiendo una cuantificación según niveles o grados con una significación de riesgo; iii.

El test de mutaciones de ADN en las heces tiene falsos positivos y falsos negativos, que varían con gran amplitud, según los autores; iv. La positividad del test de sangre oculta en las heces tiene un porcentaje elevado de falsos positivos, así como los falsos negativos no son despreciables; v. Cuando un tumor sangra, esto significa que la barrera mucosa ha sido ya destruida con erosión/invasión de la sub-mucosa, con sus vasos sanguíneos y linfáticos, no garantizando seguridad en cuanto a la metastizacion e invasión ganglionar, o sea, el tumor no es un Tis/0; vi. Las mutaciones en los tests de ADN en las heces pueden ser de otros órganos (árbol respiratorio, aparato digestivo, a montante del colorecto, etc.), no permitiendo la garantía de la localización de un eventual tumor; vii. Además, las mutaciones en los tests de ADN en las heces pueden ser fenotipos no mutantes, lo que contribuye para los abundantes falsos positivos; viii. El test de conteo de ribosomas libres, al que correspondan niveles significativamente aumentados del número de ribosomas libres en los colonócitos aislados de las heces, representa la existencia de un verdadero aumento de la síntesis descontrolada de las proteínas, no pudiendo, por eso existir falsos positivos, porque un tumor depende esencialmente de una fabricación exagerada de proteínas en el proceso de crecimiento, y eso en la célula solo puede ser hecho por los ribosomas libres;

Potencialidades de utilización clínica del RIBOGRAMA en rastreo, diagnóstico y “follow up” del cáncer del colorecto: 1. En un escenario de utilización del RIBOGRAMA, como expresión del comportamiento fenotípico de los coloncitos (densidad de ribosomas libres expresos en unidades por volumen, a lo largo del tiempo), se abrirán las puertas a muchos campos de investigación clínica y laboratorial. Permitiría el rastreo y el diagnóstico precoz del CÁNCER COLORRECTAL en condiciones propicias para poder convencer los familiares a someterse también a un test no invasivo, que no obliga a nadie a manipular sus heces y a convencerlos, en caso de curvas elevadas de RIBOGRAMA, a someterse, muy justificadamente, a una colonoscopia diagnostico-terapéutica, una vez que no hay señales clínicas; 103

RIBOGRAMA PROJECT 2. A través del RIBOGRAMA de las células descamadas de la mucosa colorrectal, vehiculadas en las heces y recogidas en un contenedor apropiado y específico, sin necesidad del paciente manipular sus heces, mediante un diseño creado y adecuado para tal finalidad, las personas tendrían un medio sencillo para adhesión a este medio de diagnóstico y rastreo generalizado y podrían ser orientadas hacia un examen endoscópico y ser encaminadas hacia un grupo de riesgo, así como ser observados y estudiados los perfiles gráficos posteriores de los valores numéricos de las cantidades ribosomas libres (RIBOGRAMA) comparativamente a una curva-mediana-padrón trazada con el estudio de grandes grupos poblacionales.

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10. Chou, I. N., O´Donnell, P. H. Black and R.O Robbin (1977), Cell density-dependent secretion of plasminogen activator by 3T3 cells. J. Cell. Physiol., 91, 31 – 32.

11. Hanahan D., Weinberg R.A. (2000) The hallmarks of cancer. Cell, 100, 57 – 70.

12. De Young L.M., Argyris T.S., Gordon G.B.(1977) Epidermal ribosome accumulation during two-stage skin tumorigenesis. Cancer Research, 37, 388 – 393.

13. Chu T.Y., Hwang K.S., Yu M.H., Lee H.S., Lai H.C., Liu J.Y. (2002) A research-based tumour tissue bank of gynaecologic oncology: characteristics of nucleic acids extracted from normal and tumour tissues from different sites. International Journal of Gynaecological Cancer, 12, 171 – 176.

14. Boon K. et al. (2001) N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis. EMBO Journal, 20, 1383 – 1393.

15. Linaggar A.K., Kemp B.L., Sneige N., Hurr K.G., Steck K., Tu Z.N., Pritsche H.A., Singletary S.E., Balch C.M. (1996) Bivariate ARN and ADN content analysis in breast carcinoma: biological significance of ARN content. Clinical Cancer Research, 2, 419 – 426.

16. Lourenco G., Embury S., Schrier S.L., Kedes L.H. (1978). Decreased ribosomal ARN content and in vitro ARN synthesis in purified bone marrow erythoblasts of patiens with idiopathic ineffective erythropoiesis and DiGuglielmo disease. America Journal of Hematology, 5, 169 – 182.

17. Johnson L.F., Abelson H.T., Green H. and Penman S. (1974) Changes in ARN relation to growth of the fibroblast I. Amounts of mARN, rARN, and tARN in resting and growing cells. Cell, 1, 95 – 100.

18. Loeb J.N. and Yeung L.L. (1975) Synthesis and degradation of ribosomal ARN in regenerating liver. J Exptl Med, 142, 575 – 587.

19. Malt R.A. and LeMaitre D.A. (1968) Turnover of ARN in the renoprival kidney. Am J Physiol, 214, 1041 – 1047.

20. Tata J.H. (1967) .The formation and distribution of ribosomes during hormone-induced growth and development. Biochem J, 104, 1-16.

21. Levin D.L., Devessa S.S., Godwin J.D. and Silverman D.T. (1974) Cancer rates and risks. Washington D.C: United States Department of Health Education and Welfare.

22. Blobel G. and Potter V.R. (1966) Nuclei from rat liver: isolation method that combines purity with high yield. Science, 154, 1662.

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RIBOGRAMA PROJECT 23. Blobel G. and Potter V.R. (1967) Studies on free and membrane-bound ribosomes in rat liver. J Mol Biol, 26, 279.

24. Raick A.N. (1974) Cell diferentiation and tumour-promotor action in skin carcinogenesis. Cancer Research, 34, 2915 – 2925.

25. De Young, Lawrence M., Argyris, Thomas S. and Gordon, Gerald B., (1977), Epidermal ribosome accumulation during two-stage skin tumorigenesis, Cancer Research 37, 388-393.

26. Ruggero, D. et al. Dyskeratosis congenita and cancer in mice deficient in ribosomal ARN modification.

Science 299, 259-262 (2003).

27. Draptchinskaia, N. et al. The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia. Nature Genet. 21, 169-175 (1999).

28. Pardee, A. B. G1 events and regulation of cell proliferation, Science 246, 603-608 (1989).

29. Pyronnet, S. & Sonenberg, N. Cell-cycle-dependent translational control, Curr. Opin. Genet. Dev. 11, 13-18 (2001).

30. Grummt, I. Regulation of mammalian ribosomal gene transcription by ARN polymerase I. Prog.

Nucleic Acid Res. Mol. Biol. 62, 109-154 (1999).

31. Grummt, I., Smith, V.A. & Grummt, F. Amino acid starvation affects the initiation frequency of nuclear ARN polymerase. Cell 7, 439- 445 (1976).

32. Kief, D. R. & Warmer, J.R. Coordinate control of syntheses of ribosomal ribonucleic acid and ribosomal proteins during nutritional shift-up in Saccharomyces cerevisiae. Mol. Cell. Biol. 1, 10071015 (1981).

33. Klein, J. & Grummt, I. Cell cycle-dependent regulation of ARN polymerase I transcription: the nucleolar transcription factor UBF is inactive in mitosis and early G1. Proc. Natl. Acad. Sci. USA 96, 6096-6101 (1999).

34. Cavanaugh, A.H. et al. Activity of ARN polymerase I transcription factor UBF blocked by gene product.

Nature 9, 177-180 (1995).

35. Beckmann, H., Chen, J.L., O’Brien, T. &Tjian, R. Coactivator and promoter-selective properties of ARN polymerase I TAFs. Science 270, 1506-1509 (1995).

36. Brandenburguer, Y., Jenkins, A., Autelitano, D. J. & Hannan, R. D. Increased expression of UBF is a critical determinant for rARN synthesis and hypertrophic growth of cardiac myocytes Faseb J. 15, 2051-2053 (2001).

37. Voit, R., Hoffmann, M. & Grummt, I. Phosphorylation by G1-specific cdk-cyclin complexes activates the nucleolar transcription factor UBF. Embo J. 18, 1891-1899 (1999).

38. Voit, R. & Grummt, I. Phosphorylation of UBF at serine 388 is required for interaction with ARN polymerase I and activation of rADN transcription. Proc. Natl. Acad. Sci. USA 98, 13631-13636 (2001).

39. Voit, R. et al. The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the Cterminal hyperacidic tail which is essential for transactivation. Embo J. 11, 2211-2218 (1992).

40. Johnson, L. F. Williams, J. G., Aberlson, H. T., Green, H. & Penman, S. Changes in ARN in relation to growth of the fibroblast. III. Posttranscriptional regulation of mARN formation in resting and growing cells. Cell 4, 69-75 (1975).

41. Baxter, G. C. & Stanners, C.P. The effect of protein degradation on cellular growth characteristic. J. Cell. Physiol. 96, 139-145 (1978).

42. Jefferies, H. B. et al. Rapamycin suppresses 5’TOP mARN translation through inhibition of p70s6k.

Embr J. 16, 3693-3704 (1997).

43. Kawasome, H. et al. Targeted disruption of p70 (s6k) defines its role in protein synthesis and rapamycin sensitivity. Proc. Natl Acad. Sci. USA 95, 5033-5038 (1998).

44. Orfao A, Ciudad J, López A, López-Berges MC, Vidriales B, Macedo A, et al.. La citometria de flujo en el diagnóstico clínico. Servicio General de Citometria. Universidad de Salamanca, 1993.

45. Orfao A, González M, Ciudad J, López-Berges MC, López A, San Miguel JF, et al. Aplicaciones de la citometría de flujo en el diagnóstico hematológico. Biol Clin Hematol 1992; 13: 456-523.

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ANEXOS TABLA I Nº de caso

1 2 3

4

5 6 7 8

Descripción cuantitativa de los ribosomas libres

Microscopia electrónica

Diagnóstico/ Información

Citoplasma filled Carcinoma pappilar numerous Numerous free invasivo, organelos, ribosomes moderadamente mitochondrias, free diferenciado ribosomes, short Free ribosomes Small clusters of free Glial C6 Cells randomly dispersed ribosomes transformadas Human Free ribosomes adrenocortical --------------------exceedingly abundant carcinoma derived SW-13 cell lines Rich a rough endoplasmic Signet ring cell reticulum (which carcinoma (SRC) Lack of free ribosomes suggested SRC cell and mucinous has a strong adenocarcinoma of capability of protein the stomach and mucus synthesis Process of carcinogenesis Increase in the content endoplasmic induced by 4 of free ribosomes dimethylaminobenze ne Friend Free ribosomes -------------------------- erythroleukemia cell growth EBV – Related liver Numerous free Some rough tumor (occurring ribosomes and endoplasmatic after kidney polysomes reticulum transplant) HEL cells – human Increase of free --------------------erythroleukemia ribosome numbers …induced

107

Referencia bibliográfica (en la TABLA siguiente) 1

2 3

4

5

6

7

8

RIBOGRAMA PROJECT

9

10

11

12

Membranes of rough endoplasmic “Principal” cytoplasmic reticulum and Golgi organelles were free apparatus were ribosomes into rosettes poorly developed in most neoplastic cell Rich in organelles of Rich in ...free this type including ribosomes microtubules … mitochondria Enlarged mitochondria Numerous free proeminent smooth ribosomes and rough endoplasmic reticulum Numerous free ribosomes

13

Increase in membrane free ribosomes

14

…cytoplasmic…mainly on free ribosomes

15

... abundant free ribosomes

16

Cytoplasm may … contain free ribosomes

17

… predominantly free ribosomes

18

Free ribosomes were present

----------------------Functional alterations… paralleled by an increase in membrane – free ribosomes…

Weakly … rough surface endoplasmic reticula cisternae and Golgi complexes After 2 to 3 weeks of ATRA treatment ER and free ribosomes became rare as the maturation process Few mitochondria, a dilated rough endoplasmic reticulum Inconspicuous rough -endoplasmic reticulum -small number of mitochondria -developed Golgi complexes Mitochondria … endoplasmic reticulum and Golgi apparatus were present 108

Bronchogenic carcinoma

9

Oligodendroglioma

10

Mammary Paget Disease

11

Intestinal tumor induced by 1,2 dimethylhydrazine

12

Rat liver hepatocarcinogenesi s induced by 0,25% DL – ethionine [hepatoma]

13

Neuroblastoma cell lines

14

Acute promielocytic leukaemia cells

15

Metastatic (neck lymph node) malignant extrarenal rhabdoid tumor

16

Ewing’s sarcoma

17

Colorectal polyps

18

RIBOGRAMA PROJECT

19 20

…exhibited greater activity (free ribosomes) Turnover of … free ribosomes was greater in host livers

21

Free ribosomes were the most prominent cellular organelles

22

Disaggregation of free and membrane bound polyribosomes

23

Free ribosomes

24

Prominent ultrastructural features… being free ribosomes

25

Prominent free ribosomes

26

Accumulation of numerous free ribosomes

27

Free ribosomes

28

Numerous free ribosomes

Hepatoma transplantable

19

Transplantable hepatoma rat cells

20

Moderate number of mitochondria, were randomly distributed.

Poorly developed granular endoplasmatic reticulum

Nasopharyngeal carcinoma

21

----------------------

Effect of aflatoxin B1 on hepatic polyribosomes and protein synthesis’ in the rat

22

Few organelles, small number of mitochondria, poor developed Golgi Keratin – positive complexes Ewing’s sarcoma inconspicuous rough endoplasmic reticulum Prominent …

Human - rough endoplasmic adrenocortical reticulum cisternal - carcinoma – derived mitochondria SW-13 cell line Thalamic tumor in a patient with human Prominent rough T-cell lymphotropic endoplasmic virus type 1 (HTLVreticulum 1) [extranodal lymphoma of the skull] Marcadly increased Neuroblastoma Bvesicular trafficking 104 cells the Golgi Cytoplasmic organelles such as Carcinoma of the rough endoplasmic thyroid gland reticulum, mitochondria Abundance of dilated rough Small cell carcinoma endoplasmic of the ovary reticulum 109

23

24

25

26

27

28

RIBOGRAMA PROJECT

29

Abundant free ribosomes

-Absent smooth endoplasmic reticulum

Carcinoma of the endometrium

29

Adrenocortical oncocytoma

30

30

Abundant free ribosomes

Abundant rough endoplasmic reticulum cisternae Smooth endoplasmic reticulum absent

31

Abundant free ribosomes

Prominent nucleoli

Chemically induced mouse hepatoblastoma

31

32

Numerous free ribosomes

Small amounts of rough endoplasmic reticulum and round mitochondria

Malignant fibrous histiocytoma

32

33

Numerous (?) free ribosomes

Numerous small mitochondria

34

Increase of free ribosomes

35

Many free ribosomes

36

Free ribosomes were common

37

Increased free ribosomes

38 39

High contents of free ribosomes Clusters of free ribosomes

40

Many free ribosomes

41

Numerous free ribosomes

Adenocarcinoma cells of an ovarian clear cell tumor Histogenesis of pseudoductular changes in pancreas of guinea pigs with N-methyl, NNitrosourea

Prominent nucleoli, marked decrease in rough endoplasmic reticulum; abundant microphilaments and microtubules Large nuclei with prominent nucleoli; small quantities of NCI â&#x20AC;&#x201C; H295R cells rough and smooth endoplasmic reticulum -Rich mitochondria - Some rough Leiomyoblastoma of endoplasmic the uterus reticulum Membrane bound ribosomes Liver tumor rats decreased -----------------------

Sarcoma 45

Dilated endoplasmic Cortical dendritic reticulum profiles spines Many rough Pulmonary clear cell endoplasmic reticuli carcinoma Hepatocarcinogenes ----------------------is induced by ethionine

110

33

34

35

36

37 38 39 40 41

RIBOGRAMA PROJECT

42

...Contain a few cytoplasmic organelles ...except for free ribosomes…

43

…tumor cells …contains free ribosomes …

Contain few cytoplasmic organelles … and a small number of mitochondria… …contain filamentous structures

44

Abundant free ribosomes

Abundant mitochondria

45 46 47

48 49 50

51

52 53

Human germ cell tumor – derived cell line Embryonal rhabdomyosarcoma in bucal mucosa…

Oncocytic metaplasia and carcinoma of the endometrium

...Referência a …referência a …“free -Golgi complex Tapioca melanoma ribosomes”…

-mitochondria of the iris...

-RER Rough endoplasmic “Lipomatous” lesions …free ribosomes… reticulum were in the liver of were present present B6C3F1 mice …numerous Pituitary distended rough … numerous free fibrosarcoma endoplasmic ribosomes following reticula, Golgi radiotherapy apparatus Abundant mitochondria, Epithelioid malignant Abundant free lysosomes, rough schwannoma cell ribosomes surface endoplasmic line reticulum … free ribosomes were …desmosomes Rat bladder tumor observed present… cell lines Nuclear lobulation Human …increases… of free and nucleolar erythroleukemia ribosomes content cells Some mitochondria and other poorly developed Numerous free cytoplasmic Rat malignant ribosomes organelles, fibrous histiocytoma suggesting undifferentiated nature Rat colorectal epithelium tumors Many free ribosomes ----------------------after treatment with carcinogen Poorly differentiated Numerous free Small cell cytoplasm numerous ribosomes osteosarcoma mitochondria 111

42

43

44

45

46

47

48

49 50

51

52

53

RIBOGRAMA PROJECT Abundance of free ribosomes

Paucity of organelles

Ewing’s sarcoma

54

55

Numerous free ribosomes

Little rough endoplasmic reticulum; few mitochondria; small Golgi complex

Mouse mammary gland adenocarcinomas

55

56

Great number of free ribosomes

-----------------------

Malignant lymphoma

56

54

57 58 59 60

61 62

63

64 65

Well developed Large amount of free Golgi apparatus, Monocytic leukaemia ribosomes rough endoplasmic cells reticula Rough endoplasmic Poorly differentiated reticulum Free ribosomes Rhabdomyosarcoma pleomorphic s mitochondria Lymphocytic … 805 free ribosomes ----------------------leukaemia tumor Prominent Golgi apparatus numerous Adenocarcinoma Clusters of free profiles of rough from apocrine ribosomes endoplasmic glands… in dogs reticulum Poor developed Golgi apparatus Myeloma cells in Few free ribosomes phenotypic features human Hybridoma characteristics for myeloma cells Few granular Numerous free Human endoplasmic ribosomes osteosarcoma reticulum Numerous organelles – many lysosomes, swollen mitochondria, Golgi Subependymal giant Many free ribosomes complexes, rough cell tumor and smooth endoplasmic reticulum Rough endoplasmic Moderate number of reticulum; Golgi Choroid plexus free ribosomes complexes, carcinoma microtubules Mitochondria and Free ribosomes more Papillary thyroid Golgi complex frequent carcinoma declined

112

57

58 59

60

61

62

63

64

65

RIBOGRAMA PROJECT

66

Free ribosomes

67

…a number of free ribosomes…

68

Free ribosomes

69

Free ribosomes

70

Free ribosomes

71

Moderate free ribosomes

72

Abundant free ribosomes

73

Free ribosomes

Reduced number of cytoplasmic organelles, Carcinomas of lung represented mainly (large cell) by mitochondria, rare Golgi vesicles and flattened tubules Friend …a number of erythroleukemia cell nucleoli growth - Pronounced smooth endoplasmic reticulum Hodgkin’s disease - Mitochondria - Nucleoli enlarged Rough endoplasmic reticulum, Golgi Rat ascitis hepatoma apparatus, smooth cells membranous vesicles Increase endoplasmic Rat ascites reticulum; some hepatoma cells mitochondria Moderate amount of rough endoplasmic reticulum, Astroblastoma mitochondria…scant y microtubules.

Prominent nucleoli Brain tumor in dogs Some rough induction by Schmidt endoplasmic – Ruppin Strain of reticulum and Rous- Sarcoma mitochondria Virus Ectopic endometrial glandular epithelium Rough endoplasmic in 5 cases of reticulum adenomyosis with mitochondria and reference to normal microfibillar proliferative structures endometrium and endometrial adenocarcinoma

113

66

67

68

69

70

71

72

73

RIBOGRAMA PROJECT

74

75

76

77 78 79 80 81 82 83 84 85

Flattened elements of rough endoplasmic Abundant free reticulum, small Vaginal ribosomes Golgi apparatus, hemangiopericytoma round or elongated mitochondria, lysosomes Desmosomes, endoplasmic reticulum, lysosomes, paucity Clear cell Free ribosomes of cytoplasmic odontogenic tumor organelles with prominent vacuolization Small nucleoli, moderate amounts Pancreatic islet cell of rough Many free ribosomes tumor causing endoplasmic acromegaly reticulum, some large Golgi Low protein content Sparse free ribosomes in cytosol, paucity of Cell condrosarcoma organelles (scanty) Few organelles, Numerous free Testicular RER, mitochondria ribosomes seminomas and Golgi apparatus Desmosomes; polymorphous Sphenoidal ridge Free ribosomes mitochondria; meningioma enlarged Golgi apparatus Differentiated Increased free Increased medullary carcinoma ribosomes polysomes of the thyroid Osteosarcoma cell Located free ribosomes Few organelles line (ROS 17/2) Increased amounts of Cutaneous -------------------------free ribosomes neurofibromas Abundant free Sparse cytoplasmic Mouse ribosomes organelles hepatoblastoma Small free ribosomes, locked Dispersed free Malignant fibrous diverse fused ribosomes histiocytoma lysosomes; RER fragmented Rat Rough endoplasmic Free ribosomes pheochromocytoma reticulum cell 114

74

75

76

77 78

79

80 81 82 83

84

85

RIBOGRAMA PROJECT

86 87 88 89 90 91 92 93

94 95 96

97

Rough endoplasmic; Clear cell; carcinoma mitochondria; Golgi of the endometrium apparatus R and T cells in High density of free head and neck ribosomes -------------------------carcinomas Rough endoplasmic Myelogenous Free ribosomes reticulum; leukaemia cell line mitochondria Golgi complexes Seminoma and (significant parathyroid Free ribosomes amounts). Rough adenoma in a snow endoplasmic leopard reticulum Numerous rough Numerous free Phylloides tumor of endoplasmic ribosomes the prostate reticulum Numerous Numerous face Malignant choroids endoplasmic ribosomes plexus pappiloma reticulum Rough endoplasmic Human A lot of free ribosomes reticulum poorly neuroblastoma cell developed line One or more prominent nucleoli; Chemically induced Abundant free small mitochondria. mouse ribosomes Occasionally hepatoblastoma desmosomes Small amounts of rough endoplasmic Malignant fibrous Numerous free reticulum and histiocytoma in a ribosomes scattered round Djungarian Hamster mitochondria Free ribosomes ------------------------Myeloma cells Mitochondria; short cisterns of granular Small clusters of free endoplasmic Transformed C6 glial ribosomes reticulum; stacks of cells in cultures Golgi complexes; pinocytic vesicles One or two nucleoli; prominent rough endoplasmic Congenital fibro Free ribosomes reticulum; well sarcoma developed Golgi apparatus Free ribosomes

115

86 87 88

89

90 91 92

93

94

95

96

97

RIBOGRAMA PROJECT

98

99

100

101

102

103

104

105

Mitochondria; numerous rough endoplasmic Human Free ribosomes reticulum; well adrenocortical exceedingly abundant developed Golgi carcinoma derivated apparatus. No SW-13 smooth endoplasmic reticulum Signet ring cell Scattered RER and carcinoma and Rich in free ribosomes fewer Golgi complex mucinous and lysosomes adenocarcinoma of the stomach Golgi apparatus; mitochondria, and microtubules were Clusters of free Human tumoral widely distributed in ribosomes ependymal cell lines cytoplasm; granular endoplasmic reticulum Abundant Poorly differentiated Abundant free cytoplasmic (“insular”) carcinoma ribosomes organelles of the thyroid gland Abundant mitochondria; dilated Ductal empty rough Free ribosomes adenocarcinoma of endoplasmic the pancreas reticulum cisternae (moderate amount) Increase Human Apparent increase free mitochondrial erythroleukemia ribosome number number cells Rough endoplasmic reticulum (RER); numerous mitochondria; Golgi Stimulatory effects area and the …of primary Many free ribosomes vesicles are osteoblast like cells numerous; … cytoskeleton consisted of many microfilaments Human pituitary Many mitochondria; tumor - derived multiple nucleoli; few follicle stellate cell Innumerable free cisternae of rough line ribosomes endoplasmic reticulum; many lysosomes 116

98

99

100

101

102

103

104

105

RIBOGRAMA PROJECT

106

107 108

109

110

111 112

113

114

115

Nucleoli are rareâ&#x20AC;Ś not rich in organelles; Carcinoma rete Show ribosomes mitochondria; rough testis endoplasmic reticulum; Golgi bodies.

Paucity cytoplasmic Abundant free Ductal papillary organelles; altered ribosomes adenocarcinoma ribosomes Moderate number of mitochondria; Numerous free Small cell carcinoma variable amounts of ribosomes of ovary paranuclear whorls of filaments Numerous microtubulus and Rich in free ribosomes mitochondria Oligodendroglioma prominent Golgi apparatus Numerous lysosomes; enlarged mitochondria; Numerous free prominent smooth Mammary Paget ribosomes and rough disease endoplasmic reticulum, tonofilaments More numerous free Adenocarcinomas of -------------------------ribosomes stomach Turnover of MRNA of Transplantable free polyribosomes is ------------------------- hepatomas and host greater liver of rats Deprived of organelles.

Human Free ribosomesâ&#x20AC;Ś Prominent rough adrenocortical prominent endoplasmic carcinoma derived reticulum cisternae SW-13 cell line and mitochondriae Protein synthesizing Free ribosomes -------------------------cell apparatus in malignant growth Small quantities of rough endoplasmic reticulum; prominent NCI-H295R cells Many free ribosomes nucleoli; numerous (human adrenal cell elongated lines) mitochondria, a few complexes 117

106

107

108

109

110

111 112

113

114

115

RIBOGRAMA PROJECT

116

117

118

119 120 121 122 123 124 125 126 127

Histogenesis of Prominent nucleoli; pseudo-ductular marked decrease in changes in pancreas Increase of free rough endoplasmic of guinea pigs ribosomes reticulum; abundant treated with Nmicrofilaments and methyl â&#x20AC;&#x201C; Nmicrotubules Nitrosourea Malignant effusions Mesothelioma cell has of pleural fewer free ribosomes mesothelioma and than adenocarcinoma -------------------------pulmonary cell adenocarcinoma Oncocytic Abundant free Abundant metaplasia and ribosomes mitochondria carcinoma of the endometrium Smooth endoplasmic reticulum virtually Adrenocortical Abundant ribosomes absent; abundant oncocytoma rough endoplasmic reticulum cisternae Dilated rough Numerous free Small cell carcinoma endoplasmatic ribosomes of the ovary reticulum Golgi apparatus Numerous free Neuroblastoma B(increase vesicular ribosomes 104 cells trafficking) Prominent rough Cerebral small cell Free ribosomes endoplasmatic tumor, in extranodal reticulum lymphoma Embryonal Filamentous rhabdomyosarcoma Free ribosomes structures â&#x20AC;Ś in the bucal mucosa Pluripotent human Few cytoplasmic Many free ribosomes germ cell tumor â&#x20AC;&#x201C; organelles derivated cell line Rat Prominent smooth Numerous free hepatocarcinogenesi endoplasmic ribosomes s induced by reticulum ethionine Many rough Pulmonary clear cell Many free ribosomes endoplasmic reticuli carcinoma Numerous free Numerous Small cell ribosomes mitochondria osteosarcoma

118

116

117

118

119

120 121 122

123

124

125 126 127

RIBOGRAMA PROJECT

128

129

130

131

132 133 134

135

136

137

Rat colorectal epithelium and Many mitochondria tumors after Many free ribosomes many collagen – treatment with Nliketonofilaments methyl – N – nitro – N – Nitrosoguanidine Some mitochondria and other poorly Transplantable rat Numerous free developed malignant fibrous ribosomes cytoplasmic histiocytoma organelles abundant mitochondria, Epithelioid malignant Abundant free lysosomes and schwannoma cell ribosomes rough surfaced line (YST-1) endoplasmic reticulum Numerous distended rough endoplasmic Numerous free reticula, Golgi Pituitary adenoma ribosomes apparatus. Some desmosome – like.

Existence of Human microtubules; rough A lot of free ribosomes neuroblastoma cell endoplasmic are line (HSNB) poorly developed Large malignant Numerous free Rough endoplasmic choroids plexus ribosomas reticulum pappiloma in the third ventricle Numerous Numerous free mitochondria, Golgi Central neurocytoma ribosomes apparatus.

Microtubules Significant amounts Seminoma and of rough Significant amounts of parathyroid endoplasmic free ribosomes adenoma (snow reticulum, Golgi leopard) complexes Rough endoplasmic reticulum, Myelogenous Free ribosomes mitochondria, leukaemia cell line microfilament bundles Round mitochondria; Golgi apparatus; Clear cell carcinoma Free ribosomes rough endoplasmic of the endometrium reticulum

119

128

129

130

131

132

133

134

135

136

137

RIBOGRAMA PROJECT

138

Free ribosomes

139

Many free ribosomes

140

Abundant free ribosomes

141 142 143

144

145

146

147

Rough endoplasmic reticulum Prominent nucleoli; fragmented rough endoplasmic reticulum (RER); occasional small mitochondria Sparse cytoplasmic organelles

Rat adrenal chromaffin and pheochromocytoma cells

138

Malignant fibrous histiocytoma

139

Mouse hepatoblastoma N-methyl-NAbundant amylnitrosamina â&#x20AC;&#x201C; Free ribosomes tonofilaments and induced rat mitochondria oesophageal carcinogenesis Rich in rough Naked nuclei in Rich in free ribosomes surface endoplasmic breast aspirate reticulum smears Poorly differentiated Increased free -------------------------- medullary carcinoma ribosomes of the thyroid Scant organelles.

Tumor cells packed Numerous free with organelles Testicular ribosomes including RER, seminomas mitochondria; Golgi apparatus Small nucleoli; moderate amounts Pancreatic islet cell of rough Many free ribosomes tumor causing endoplasmatic acromegaly reticulum. Some large Golgi fields -Desmosomes -Lysosomes Clear cell Free ribosomes -Endoplasmic odontogenic tumor reticulum Flattened elements of rough endoplasmic Abundant free reticulum, small Vaginal ribosomes Golgi apparatus, hemangiopericytoma elongated mitochondria, lysosomes

120

140

141

142 143

144

145

146

147

RIBOGRAMA PROJECT

148

Abundant free ribosomes

149

Moderate amount of free ribosomes

150

Free ribosomes

151

Free ribosomes

152

Free ribosomes

153

Numerous free ribosomes

154

Moderate number of free ribosomes

155

Numerous free ribosomes

Some rough endoplasmic reticulum and mitochondria

Brain tumor induced in dogs by intracerebral inoculation of SRRSV induced cultured tumor cells

Moderate amount of rough surface endoplasmic Astroblastoma reticulum, mitochondria, scanty microtubules Rough endoplasmic Rat ascitis hepatoma reticulum; some cells mitochondria Rough endoplasmic reticulum; Golgi apparatus; smooth â&#x20AC;&#x201C; Rat ascitis hepatoma membranous cells vesicles; mitochondria Rare Golgi vesicles and flattened tubules; (large cell) mitochondria; carcinomas of the various amounts of lung tonofilaments; rough endoplasmic reticulum Little rough endoplasmic Mouse mammary reticulum; few gland mitochondria, small adenocarcinomas Golgi; abundant organelles Rough endoplasmic reticulum; microtubules; Choroids plexus numerous carcinoma mitochondria; Golgi complexes Few membranes of granular Osteosarcoma endoplasmic reticulum (ER)

121

148

149

150

151

152

153

154

155

RIBOGRAMA PROJECT

156

Clusters of free ribosomes

157 158

Great number of free ribosomes Abundance of free ribosomes

159

Increase number of free ribosomes

160

Free ribosomes

161

Increased numbers of free ribosomes

162

Abundant free ribosomes

163

Free ribosomes

164

Free ribosomes

165

Numerous free ribosomes

166

Large amount of free ribosomes

167

Abundant free ribosomes

Numerous profiles of rough endoplasmic reticulum; prominent Golgi apparatus

Adenocarcinomas derived from apocrine glands of the anal sac associated with hipercalcemic dogs

156

-------------------------

Malignant lymphoma

1577

Paucity of organelles

-------------------------

Extraskeletal Ewing`s sarcoma Ultrastructure of oesophageal epitelium in displastic foci Branchiometric paragangliomes

Abundant rough endoplasmic Pituitary adenomas reticulum -microtubules; moderate Golgi Human complexes; scant neuroblastoma lines granular SK-N-SH, SK-Nendoplasmic BE(2), and SK-N-MC reticulum Lack of SER; Postcastrational abundant RER; adrenocortical prominent Golgi carcinomas in complexes; few female CE-Mice mitochondrias Numerous mitochondria; Melanocytomas prominent rough (magnocellular nevi) endoplasmic of the optic reticulum Moderate development of the MtTw15 rough endoplasmic mamosomatrofic reticulum; prominent tumor Golgi profiles Large amount of rough endoplasmic Spontaneous reticulum; testicular neoplasm lysosomes; mitochondria Epithelial tumors induced in the rat Many mitochondria kidney by nitrosomorpholine 122

158 159 160 161

162

163

164

165

166

167

RIBOGRAMA PROJECT

169

Abundant free ribosomes Increased free ribosomes

170

Abundance of free ribosomes

168

Well developed RER Mitochondria increased Absence of smooth endoplasmic reticulum

171

Free ribosomes

Desmosomes; relative few other organelles

172

Free ribosomes

Scant organelles

173

Abundant free ribosomes

174

More numerous free ribosomes

175

Abundant free ribosomes

176

Free ribosomes

177

Free ribosomes randomly distributed

178

Very abundant free ribosomes

179

Free ribosomes

180

Numerous free ribosomes

181

Increase free ribosomes

182

Free ribosomes

Hepatoblastoma of fetal liver Uveal malignant melanoma Hepatocellular tumors Transitional cloacogenic carcinomas of anorectal region Maxillofacial synovial sarcoma

Desmosomes; rough endoplasmic Stromal sarcoma of reticulum; variable breast amounts of microfilaments Adenocarcinoma of -------------------------stomach Increased organelle content; many fine Cholangiocarcinoma fibrils Endoplasmatic Cultured hepatoma reticulum; cells mitochondria Golgi apparatus, Monoclonal Hodgkin mitochondria; fibrils cells cultured Functional mouse Smooth endoplasmic adrenal cortical reticulum tumor Numerous profiles of granular Gastric endoplasmic leiomyosarcoma reticulum Well developed Hyperplasic foci in rough endoplasmic precancerous rat reticulum; liver peroxisomes BBN (N-Butyl-N [14 hydroxybutyl] â&#x20AC;&#x201C; Microfibrils were nitrosamine â&#x20AC;&#x201C; increased induced rat bladder cancer Establishment of a -------------------------- human rectal cancer cell line

123

168 169 170

171 172

173

174 175 176 177 178

179

180

181

182

RIBOGRAMA PROJECT

183

Prominent free ribosomes

184

Free ribosomes

185

Abundant free ribosomes

186 187 188 189 190 191 192 193 194

195

Prominent mitochondria and Golgi; abundant rough endoplasmic reticulum and adjacent collagen fibrils Rough surfaced endoplasmic reticulum; prominent Golgi complex --------------------------

Malignant fibrous histiocytoma of the orbit

183

Spontaneous pituitary adenomas in aging rats

184

Pseudolymphoma of the lung

185

Rough endoplasmic Myeloma cells reticulum (RER) Golgi apparatus; Increased amounts of decreased amounts Clear cell thyroid free ribosomes of rough carcinoma endoplasmic Endoplasmatic Increase in free Hepatocarcinogenes reticulum ribosomes is membranes Salivary gland Abundant free Numerous carcinoma ribosomes desmosomes (myoepithelioma) Dilated rough Abundant free endoplasmic Epithelioid sarcoma ribosomes reticulum; prominent Golgi systems Few tubules and Metastasising Abundant free filaments, Golgi malignant epithelioid ribosomes membranes schwannoma Zajdela hepatoma Increase free ribosome ------------------------- rat liver and ascitis cells Decrease in number Numerous free Human tongue of mitochondria; ribosomes carcinoma cells tonofilaments Prominent nucleoli: short segments of Myelomonocyte Moderate number of endoplasmic leukaemia and free ribosomes reticulum; Golgi Hodgkinâ&#x20AC;&#x2122;s disease lamellae Production of intestinal and other Little RER, Numerous free tumors by 1,2 â&#x20AC;&#x201C; prominent Golgi ribosomes dimethylhydrazine bodies dihydrochloride in mice Free ribosomes

124

186 187

188 189

190

191 192 193

194

195

RIBOGRAMA PROJECT

196

Greatly increased content of free ribosomes

197

Free ribosomes

198

Marked increase in free ribosomes

199

Rich in ribosomes

200

Free ribosomes

201

Abundant free ribosomes

202

Free ribosomes

203

Abundant free ribosomes

204

Large numbers of free ribosomes

205

An abundance of free ribosomes

lysosomes Abundant mitochondria; prominent Golgiâ&#x20AC;&#x2122;s bodies Extensive endoplasmic reticulum; considerable increase of mitochondria --------------------------

Rats bearing chemically induced hepatomas Poorly differentiated adenocarcinoma of the human tuba uterinae

198

Epidermal carcinogenesis

199

Ependymoma

Sparsely rough endoplasmatic Malignant lymphoma reticulum; bizarre mitochondria Rough endoplasmic Mouse reticulum rhabdomyosarcoma

125

197

Rat epidermis during experimental carcinogenesis

Expanded Golgi complexes, Pituitary thyrotrophic abundant rough tumor endoplasmic reticulum Cytoplasm scanty; Primary Golgi apparatus well lymphosarcoma of developed; testis prominent nucleoli Abundant rough endoplasmic Dermatofibroma reticulum; bundles of (histiocytoma) filaments --------------------------

196

200

201

202 203 204 205

RIBOGRAMA PROJECT BIBLIOGRAFIA de los dados de la TABLA. 1 N.º de REF 1 2 3 4

5 6

7

8 9

10 11 12

13

14 15

16

17

18 19 20 21

22 23

DOCUMENTO Breast cancer tissues 2002 ; AccuMax Array – Isu ABXIS co. LTD Mareš V., Krajči D., Lisá V. The subcellular targets of Mercaptoborate (BSH), a carrier of B for Neutron Capture Therapy (BNCT) of brain tumors. Physiol. 2003; Res. 52: 629-635.

Albertin G, et al. Endothelin-1 and adrenomedullin enhance the growth of human adrenocortical carcinoma-derived SW-13 cell line by stimulating proliferation and inhibiting apoptosis. International Journal of Molecular Medicine 2005; 15: 469-474.

Yang, XF, et al. Pathobiological behaviour and molecular mechanism of signet ring cell carcinoma and mucinous adenocarcinoma of the stomach: a comparative study. World Journal of Gastroenterology 2004; 10: 750-754.

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26 27 28 29

30 31 32 33 34 35

36

37 38 39

40 41

42

43 44

45 46 47

48

Carraro AG, et al. Endothelin-1 and adrenomedullin enhance the growth of human adrenocortical carcinoma-derived SW-13 cell line by stimulating proliferation and inhibiting apoptosis. Int J Mol Med. 2005; 15(3): 469-474.

Komori T, et al. An unclassified cerebral small cell tumor in a patient with human T-cell lymphotropic virus type 1-induced primary extranodal lymphoma. Mod Pathol.Oct.1995; 8(8): 811-816.

Oâ&#x20AC;&#x2122;Neill BJ, et al. Ultrastructural consequences of radical damage before and after differentiation of neuroblastoma B-104 cells. Acta Neuropathol (Berl) Jul. 1996; 92(1): 75-89.

Paik SS, et al. Poorly differentiated (insular) carcinoma of the thyroid gland: two cases report.

J Korean Med Sci. Feb. 1997; 12(1): 70-74 Forster C, et al. Small cell carcinoma of the ovary, hypercalcemic type : a case report with immunohistochemical, ultrastructural and cytophotometric analysis and review of the literature. Gen Diagn Pathol. Jun. 1997; 142(5-6): 365-370.

Silver SA, Cheung AN, Tavassoli FA. Oncocytic metaplasia and carcinoma of the endometrium: an immunohistochemical and ultrastructural study. Int J Gynecol Pathol. Jan. 1999; 18(1): 12-19.

Macchi C, et al. Adrenocortical oncocytoma: case report and review of the literature. Tumori May-Jun. 1998; 84(3): 403-407.

Kobayashi K, et al. Establishment and characterization of a cell line from a chemically-induced mouse hepatoblastoma. J Vet Med Sci. Mar. 2000; 62(3): 263-267.

Endo Y, et al. Malignant fibrous histiocytoma in a Djungarian hamster. J Vet Med Sci. May 2000; 62(5): 539-541.

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Rao MS, Reddy JK. Histogenesis of pseudo-ductular changes induced in the pancreas of guinea pigs treated with N-methyl-N-nitrosourea. Carcinogenesis 1980; 1 (12): 1027-1037.

Thomopoulos GN, Kyurkchiev S, Perbal B. Immunocytochemical localization of NOVH protein and ultrastructural characteristics of NCI-H295R cells. J Submicrosc Cytol Pathol. Jul. 2001; 33(3): 251-260.

Watanabe K, Ogura G, Suzuki T. Leiomyoblastoma of the uterus: an immunohistochemical and electron microscopic study of distinctive tumours with immature smooth muscle cell differentiation mimicking fetal uterine myocytes. Histopathology Apr. 2003; 42(4): 379-386.

Sharykina NI, Kudriavtseva IG, Pavlovskaia GP. Protein synthesizing cell apparatus in malignant growth and pharmacotherapy by chlofiden. Ukr Biokhim Zh. Jul.-Aug. 2002; 74 (4): 80-84.

Castejon OJ, Castellano A, Arismendi G. Transmission electron microscopy of cortical dendritic spines in the human oedematous cerebral cortex. J Submicrosc Cytol Pathol. Apr. 2004; 36(2): 181-191.

Inase N, et al. Pulmonary clear cell carcinoma. J Surg Oncol. Oct. 1991; 48(2): 145-147.

Novikoff PM, et al. Characterizations of and interactions between bile ductule cells and hepatocytes in early stages of rat hepatocarcinogenesis induced by ethionine. Am J Pathol.

Dec. 1991; 139(6): 1351-1368.

Damjanov I, Horvat B, Gibas Z. Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, NCCIT. Lab Invest. Feb. 1993; 68(2): 220232.

Yoshihara T, Nabeshima M, Ishii T. Embryonal rhabdomyosarcoma arising in the buccal mucosa: a case report with immunohistochemical and electronmicroscopic findings. Int J Pediatr Otorhinolaryngol. Jan. 1994; 28 (2-3): 247-255.

Silver SA, Cheung AN, Tavassoli FA. Oncocytic metaplasia and carcinoma of the endometrium: an immunohistochemical and ultrastructural study. Int J Gynecol Pathol.

Jan.1999; 18 (1): 12-19.

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Dixon D, et al. Lipomatous lesions of unknown cellular origin in the liver of B6C3F1 mice. Vet Pathol Mar. 1994; 31(2): 173-182.

Sato K, et al. Clinical and histological study of pituitary fibrosarcoma following radiotherapy for pituitary adenoma: case report. Neurol Med Chir (Tokyo) 1990; 30 (11 Spec No): 888-92.

Nagashima Y, et al. Establishment of an epithelioid malignant schwannoma cell line (YST-1).

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49 50 51

52

53 54 55 56 57 58 59 60

61

62

63 64 65 66 67 68 69

70

71 72

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Nagata Y. Ultrastructure of cultured normal bladder transitional epithelial cell and BBN induced rat bladder tumor cell lines. Nippon Hinyokika Gakkai Zasshi Feb. 1990; 81(2): 188195.

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Yamate J, et al. Characteristics of cis-diamminedichloroplatinum-selected in vitro passaged cells derived from a transplantable rat malignant fibrous histiocytoma. Acta Pathol Jpn. Aug.1990; 40(8): 554-561.

Zusman I, Zimber A. Ultrastructural changes in rat colorectal epithelium and tumors after treatment with N-methyl-N’-nitro-N-nitrosoguanidine and secondary bile acids. Acta Anat (Basel) 1991; 141(3): 282-288.

Dickersin GR, Rosenberg AE. The ultrastructure of small-cell osteosarcoma, with a review of the light microscopy and differential diagnosis. Hum Pathol. Mar. 1991; 22(3): 267-275.

Berthold F, et al. Ultrastructural, biochemical, and cell-culture studies of a presumed extraskeletal Ewing’s sarcoma with special reference to differential diagnosis from neuroblastoma. J Cancer Res Clin Oncol. 1982; 103(3): 293-304.

Ames IH, et al. Fine structure analysis and surface characteristics of mouse mammary gland adenocarcinomas. Scan Electron Microsc. 1984; pt 1: 391-399 Averdunk R, et al. Development and properties of malignant lymphoma induced by magnesium deficiency in rats. J Cancer Res Oncol. 1982; 104 (1-2): 63-73.

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Traganos F, et al. Effects of retinoic acid versus dimethylsulfoxide on Friend erythroleukemia cell growth: II-Induction of quiescent, nonproliferating cells. J Natl Cancer Inst. Jul. 1984; 73 (1): 205-218.

Olsson L, et al. Establishment and characterization of a cloned giant cell line from a patient with Hodgkin’s disease. J Natl Cancer Inst. Oct. 1984; 73(4): 809-830.

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74 75 76

77 78 79 80 81 82 83 84 85 86

87 88 89 90 91

92 93 94 95 96 97 98 99

Hayata T, Kawashima Y. Ultrastructure of the ectopic endometrial glandular epithelium in five cases of adenomyosis: with particular reference to the normal proliferative endometrium and endometrial adenocarcinoma. Nippon Sanka Fujinka Gakkai Zasshi Jun. 1985; 37(6): 879887.

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Saeger W, Schulte HM, Kloppel G. Morphology of a GHRH producing pancreatic islet cell tumour causing acromegaly. Virchows Arch A Pathol Anat Histopathol. 1986; 409(4): 547554.

Ohno T, et al. Ultrastructural study of a clear cell chondrosarcoma. Ultrastruct Pathol. 1986; 10(4): 321-330.

Stiller D, Holzhausen HJ. Ultrastructural cytology of testicular seminomas. Zentrallbl Allg Pathol. 1986; 131(5): 437-439.

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Kakudo K, et al. Ultrastructural study of poorly differentiated medullary carcinoma of the thyroid. Virchows Arch A Pathol Anat Histopathol 1987; 410 (5): 455-460.

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Imai Y, et al. Malignant fibrous histiocytoma: similarities to the fibrohistiocytoid cells in chronic inflammation. Virchows Arch A Pathol Anat Histopathol. 1989; 414(4): 285-298.

Oomori Y, et al. Intracellular localization of tyrosine hydroxylase immunoreactivity in the rat adrenal chromaffin and pheochromocytoma cells. Acta Anat (Basel) 1989; 136(1): 1-8.

Yorishima M, et al. Clear cell carcinoma of the endometrium with lipid producing activity: Histologic and ultrastructural study suggesting a unique neoplasm. Int J Gynecol Pathol. 1989; 8(3): 286-295.

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Sugawara I, et al. Immunocytochemichal identification and localization of the Mr 22,000 calcium binding protein (sorcin) in an adriamycin resistant myelogenous leukemia cell line.

Jpn J Cancer Res. 1989; 80(5): 469-474.

Doster AR, Armstrong DL, Bargar TW. Seminoma and parathyroid adenoma in a snow leopard (Panthera unica). J Comp Pathol. May. 1989; 100(4): 475-480.

Ito H, et al. Phyllodes tumor of the prostate: a case report. Jpn J Clin Oncol. Sep. 1989; 19(3): 299-304.

Sato K, et al. A case of large malignant choroids plexus papilloma in the third ventricle immunohistochemical and ultrastructural studies [article in japanese]. No To Shinkei Oct. 1989; 41(10): 973-978.

Ono I, et al. Establishment and characterization of human neuroblastoma cell line (HSNB).

Hum Cell Dec.1989; 2(4): 416-422.

Kobayashi K, et al. Establishment and characterization of a cell line from a chemically induced mouse hepatoblastoma. J. Vet. Med. Sci. 1999; 62(3): 263-267.

Endo Y, et al. Malignant fibrous histiocytoma in a Djungarian Hamster. J. Vet. Med. Sci. 1999; 62(5): 539-541.

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Mareš V, Krajči D, Lisá V. The subcellular targets of mercaptoborate (BSH), a carrier of B for neutron capture therapy (BNCT) of brain tumors. Physiol. Res. 2002 ; 52: 629-635.

Natalia E, et al. Congenital fibrosarcoma : report of one case treated with pre-surgical chemotherapy. International Pediatrics 2003; vol. 18, n. 2: 87-91.

Albertin G, et al. Endothelin 1 and adrenomedullin enhance the growth of human adrenocortical carcinoma derived SW-13 cell line by stimulating proliferation and inhibiting apoptosis. International Journal of Molecular Medicine 2004; 15: 469-474.

Fei X, et al. Pathobiological behaviour and molecular mechanism of signet ring cell carcinoma

129

RIBOGRAMA PROJECT

100 101 102

103 104 105 106

107

108 109 110 111 112 113

114 115 116 117

118

119 120

121 122

123 124

and mucinous adenocarcinoma of the stomach: a comparative study. World Journal of Gastroenterology 2004; 10(5): 750-754.

Brisson C, et al. Establishment of human tumoral ependymal cell lines and coculture with tubular like human endothelial cells. International Journal of Oncology 2002; 21: 775-785.

Paik SS, Kim WS, Park MH. Poorly differentiated (insular) carcinoma of the thyroid gland: two cases report. JKMS 1996; 12: 70-74.

Joo YE. A case of osteoclast like giant cell tumor of the pancreas with ductal adenocarcinoma: histopathological, immunohistochemical, ultrastructural and molecular biological studies. J Korean Med Sci 2005; 20: 516-520.

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Gerber, et al. Stimulatory effects of creatine on metabolic activity differentiation and mineralization of primary osteoblast like cells in monolayer and micromass cell cultures.

European Cells and Materials 2005; vol. 10: 8-22.

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Sidransky H, Murty CN, Verney E. Turnover of messenger ARN in transplantable hepatomas and host liver of rats. Cancer Res. Jun. 1978; 38(6): 1645-1653.

Albertin G, et al. Endothelin-1 and adrenomedullin enhance the growth of human adrenocortical carcinoma derived SW-13 cell line by stimulating proliferation and inhibiting apoptosis. Int J Mol Med. Mar. 2005; 15(3): 469-474.

Sharykina NI, Kudriavtseva IG, Pavlovskaia GP. Protein synthesizing cell apparatus in malignant growth and pharmacotherapy by chlofiden. Ukr Biokhim Zh. Jul.-Aug. 2002; 74(4): 80-84.

Thomopoulos GN, Kyurkchiev S, Perbal B. Immunocytochemical characteristics of NCIH295R cells. J Submicrosc Cytol Pathol. Jul. 2001; 33(3): 251-260.

Rao MS, Reddy JK. Histogenesis of pseudo ductular changes induced in the pancreas of guinea pigs treated with N-methyl-N-nitrosourea. Carcinogenesis 1980; 1(12): 1027-1037.

Sakuma N, Kamei T, Ishihara T. Ultrastructure of pleural mesothelioma and pulmonary adenocarcinoma in malignant effusions as compared with reactive mesothelial cells. Acta Cytol. Sep.-Oct. 1999; 43(5): 777-785.

Silver SA, Cheung AN, Tavassoli FA. Oncocytic metaplasia and carcinoma of the endometrium: an immunohistochemical and ultrastructural study. Int J Gynecol Pathol. Jan. 1999; 18(1): 12-19.

Macchi C. Adrenocortical oncocytoma: case report and review of the literature. Tumori May.Jun. 1998; 84(3): 403-407.

Forster C, et al. Small cell carcinoma of the ovary, hypercalcemic type : a case report with immunohistochemical, ultrastructural and cytophotometric analysis and review of the literature. Gen Diagn Pathol. Jun. 1997; 142(5-6): 365-370.

Oâ&#x20AC;&#x2122;Neil BJ, et al. Ultrastructural consequences of radical damage before and after differentiation of neuroblastoma B-104 cells. Acta Neuropathol (Berlin) Jul. 1996; 92(1): 75-89.

Komori T, et al. An unclassified cerebral small cell tumor in a patient with human T-cell lymphotropic virus type 1 induced primary extranodal lymphoma. Mod Pathol. Oct. 1995; 8(8): 811-816.

Hida C, et al. Ultrastructural localization of anti Purkinje cell antibody binding sites in paraneoplastic cerebellar degeneration. Arch Neurol. Jun. 1994; 51(6): 555-558.

Yoshihara T, Nabeshima M, Ishii T. Embryonal rhabdomyosarcoma arising in the buccal mucosa: a case report with immunohistochemical and electronmicroscopic findings. Int J

130

RIBOGRAMA PROJECT

125 126

127 128 129

130 131 132 133 134 135 136 137

138

139 140 141 142

143 144 145 146 147 148 149 150 151

Pediatr Otorhinolaryngol. Jan. 1994; 28(2-3): 247-255.

Damjanov I, Horvat B, Gibas Z. Retinoic acid induced differentiation of the developmentally pluripotent human germ cell tumor derived cell line. Lab Invest. Feb. 1993; 68(2): 220-232.

Novikoff PM, et al. Characterizations of and interactions between bile ductule cells and hepatocytes in early stages of rat hepatocarcinogenesis induced by ethionine. Am J Pathol.

Dec. 1991; 139(6): 1351-1368.

Inase N, et al. Pulmonary clear cell carcinoma. J Surg. Oncol. Oct. 1991; 48(2): 145-147.

Dickersin GR, Rosenberg, AE. The ultrastructure of small cell osteosarcoma, with a review of the light microscopy and differential diagnosis. Hum Pathol. Mar. 1991; 22(3): 267-275.

Zusman I, Zimber A. Ultrastructural changes in rat colorectal epithelium and tumors after treatment with N-methyl-N’-nitro-N-nitrosoguanidine and secondary bile acids. Acta Anat (Basel) 1991; 141(3): 282-288.

Yamate J, et al. Characteristics of cis-diamminedichloroplatinum-selected in vitro passaged cells derived from a transplantable rat malignant fibrous histiocytoma. Acta Pathol Jpn. Aug. 1990; 40(8): 554-561.

Nagashima Y. Establishment of an epithelioid malignant schwannoma cell line (YST-1).

Virchows Arch B Cell Pathol. 1990; 59(5): 321-327.

Sato K, et al. Clinical and histological study of pituitary fibrosarcoma following radiotherapy for pituitary adenoma: Case report. Neurol Med Chir (Tokyo). 1990; 30(11 Spec No): 888-892.

Ono I, et al. Establishment and characterization of human neuroblastoma cell line (HSNB).

Hum cell. Dec. 1989; 2(4): 416-422.

Sato K, et al. A case of large malignant choroids plexus papilloma in the third ventricle: immunohistochemical and ultrastructural studies. No To Shinkei Oct. 1989; 41(10): 973-978.

Tsujita Y, Nagashima K, Takakura K. A clinicopathological study of central neurocytoma. No To Shinkei Jun. 1989; 41(6): 547-558.

Doster AR, Armstrong DL, Bargar TW. Seminoma and parathyroid adenoma in a snow leopard (Panthera unica). J Comp Pathol. May. 1989; 100(4): 475-480.

Sugawara I, et al. Immunocytochemical identification and localization of the Mr 22,000 calcium-binding protein (sorcin) in an adriamcyn-resistant myelogenous leukaemia cell line.

Jpn J Cancer Res. May. 1989; 80(5): 469-474.

Yorishima M, et al. Clear cell carcinoma of the endometrium with lipid-producing activity: Histologic and ultrastructural study suggesting a unique neoplasm. Int J Gynecol Pathol. 1989; 8(3):286-295.

Oomori Y, et al. Intracellular localization of tyrosine hydroxylase immunoreactivity in the rat adrenal chromaffin and pheochromocytoma cells. Acta Anat (Basel) 1989; 136(1): 1-8.

Imai Y, et al. Malignant fibrous histiocytoma: similarities to the “fibrohistiocytoid cells” in chronic inflammation. Virchows Arch A Pathol Anat Histopathol. 1989; 414(4): 285-298.

Nonoyama T, et al. Mouse hepatoblastomas: a histologic, ultrastructural, and immunohistochemical study. Vet Pathol. Jul. 1988; 25(4): 286-296.

Kuwayama H, Eastwood GL. Light and electron microscopic and autoradiographic studies on N-methyl-N-amylnitrosamine-induced rat esophageal carcinogenesis. Dig Dis Sci. Jan. 1988; 33(1):83-91.

Tsuchiya S, et al. Cytologic characteristics and origin of naked nuclei in breast aspirate smears. Acta Cytol. May-Jun. 1987; 31(3): 285-290.

Kakudo K, et al. Ultrastructural study of poorly differentiated medullary carcinoma of the thyroid. Virchows Arch A Pathol Anat Histopathol. 1987; 410(5): 455-460.

Stiller D, Holzhausen HJ. Ultrastructural cytology of testicular seminomas. Zentralbl Allg Pathol. 1986; 131(5): 437-449.

Saeger W, Schulte HM, Kloppel G. Morphology of a GHRH producing pancreatic islet cell tumour causing acromegaly. Virchows Arch A Pathol Anat Histopathol. 1986; 409(4): 547-554.

Eversole LR, Belton CM, Hansen LS. Clear cell odontogenic tumor : histochemical and ultrastructural features. J Oral Pathol. Sep.1985; 14(8): 603-614.

Hiura, et al. Vaginal hemangiopericytoma: a light microscopic and ultrastructural study.

Gynecol Oncol. Jul.1985; 21(3): 376-384.

Shimura T, et al. Brain tumor induced in dogs by intracerebral inoculation of SR-RSV induced cultured tumor cells: electron microscopic study. No Shinkei Geka May. 1985; 13(5): 521-528.

Kubota, et al. The fine structure of astroblastoma. Cancer Feb. 1985; 55(4): 745-750.

Torii M, Miyake K, Kanai K. Effects of dibutyryl adenosine 3’-5’ cyclic monophosphate on the ultrastructure of rat ascites hepatoma cells and on the intracellular localization of alphafetoprotein. Gann Dec. 1984; 75(12): 1083-1088.

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Hattori R. An immunoelectron microscopic study on the distribution of a cell surfaceassociated adhesive glycoprotein synthesized by rat ascites hepatoma cells. J Cell Sci. Oct. 1984; 71: 95-109.

Nicolescu PG, Eskenasy A. Ultrastructure of macrocellular (large cell) carcinomas of the lung.

Morphol Embryol (Bucur.) Jul.-Sep.; 30(3): 211-216.

Ames IH. Fine structure analysis and surface characteristics of mouse mammary gland adenocarcinomas. Scan Electron Microsc. 1984; (Pt 1): 391-399.

155

McComb RD, Burger PC. Choroid plexus carcinoma: report of a case with immunohistochemical and ultrastructural observations. Cancer Feb. 1983; 51(3): 470-475.

156

Roessner A, et al. Biologic characterization of human bone tumours: II-Distribution of different collagen types in osteosarcoma: a combined histologic, immunofluorescence and electron microscopic study. J Cancer Res Clin Oncol. 1983; 106(3): 234-239.

157

Meuten DJ. Ultrastructural evaluation of adenocarcinomas derived from apocrine glands of the anal sac associated with hypercalcemia in dogs. Am J Pathol. May. 1982; 107(2): 167-175.

158

Averdunk R, et al. Development and properties of malignant lymphoma induced by magnesium deficiency in rats. J Cancer Res Clin Oncol. 1982; 104(1-2): 63-73.

Berthold F, et al. Ultrastructural, biochemical, and cell-culture studies of a presumed extraskeletal Ewing’s sarcoma with special reference to differential diagnosis from neuroblastoma. J Cancer Res Clin Oncol. 1982; 103(3): 293-304.

Kolycheva NI, et al. Ultrastructure of esophageal epithelium under normal conditions and in dysplastic foci. Arkh Patol. 1982; 44(2): 34-41.

Stiller D, et al. Investigations on the ultrastructural cytology of branchiomeric paragangliomas.

Anat Anz. 1982; 151(1): 50-63.

159 160 161 162 163 164 165 166

167 168

169 170 171 172 173 174 175

Saeger W, Ludecke DK. Pituitary adenomas with hyperfunction of TSH : frequency, histological classification, immunocytochemistry and ultrastructure. Virchows Arch A Pathol Anat Histol. 1982; 394(3): 255-267.

Barnes EN, et al. The fine structure of continuous human neuroblastoma lines SK-N-SH, SKN-BE (2), and SK-N-MC. In Vitro, Jul. 1981; 17(7): 619-631.

Sharawy MM, et al. Fine structural study of postcastrational adrenocortical carcinomas in female CE-mice. Anat Rec. Sep. 1980; 198(1): 125-133.

Juarez CP, Tso MO. An ultrastructural study of melanocytomas (magnocellular nevi) of the optic disk and uvea. Am J Ophthalmol. Jul. 1980; 90(1): 48-62.

Parsons JA, Baskin DG, Erlandsen SL. Heterogeneity of the MtTw15 mammosomatotropic tumour: II – Characterization of parenchymal cells by superimposition immunocytochemistry and electron microscopy. Anat Rec. Mar. 1980; 196(3):301-311.

Chung KW. Spontaneous testicular neoplasm in mice with testicular feminization. Cell Tissue Res. 1980; 208(1): 46-56.

Bannasch P, Krech R, Zerban H. Morphogenesis and micromorphology of epithelial tumors induced in the rat kidney by nitrosomorpholine. IV. Tubular lesions and basophilic tumors (author’s transl.). J Cancer Res Clin Oncol. 1980; 98(3): 243-265.

Horie A, Kotoo Y, Hayashi I. Ultrastructural comparison of hepatoblastoma and hepatocellular carcinoma. Cancer Dec. 1979; 44(6): 2184-2193.

Mincione GP, Campana G, Vannelli G. Study of uveal malignant melanoma with the electrón microscope. J Fr Ophtalmol. Oct. 1979; 2(10): 531-538.

Hruban Z. Ultrastructure of hepatocellular tumors. J Toxicol Environ Health. Mar-May 1979; 5(2-3): 403-433.

Otto HF, Gebbers JO, Winkler R. Ultrastructural observations on transitional cloacogenic carcinomas of the ano-rectal region (author’s transl.). Virchows Arch A Pathol Anat Histol.

Feb. 1979; 381(2): 223-239.

Nunez Alonso C, Gashti EN, Christ ML. Maxillofacial synovial sarcoma : light and electron microscopic study of two cases. Am J Surg Pathol. Feb. 1979; 3(1): 23-30.

Tang PH, Petrelli M, Robechek PJ. Stromal sarcoma of breast: a light and electron microscopic study. Cancer Jan. 1979; 43(1): 209-217.

Dikshtein EA, et al. Degree of maturity and histogenesis of stomach cancer and their importance for prognosis. Arkh Patol. 1979; 41(7): 25-32.

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RIBOGRAMA PROJECT 176 177 178

179 180 181 182

183 184 185 186 187 188 189 190 191

192 193 194 195 196

197 198 199 200

201 202

Stitnimankarn T, et al. Ultrastructure of cholangiocarcinoma associated with opisthorchiasis.

Southeast Asian J Trop Med Public Health Dec. 1978; 9(4): 558-567.

Lopez Saura P, Trouet A, Tulkens P. Analytical fractionation of cultured hepatoma cells (HTC cells). Biochim Biophys Acta. Nov. 1978; 543(4): 430-449.

Schmid EN, Boecker WR, Lickfeld KG. The fine structure of monoclonal Hodgkin cells cultured in diffusion chambers. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol. 30 Oct. 1978; 92(3): 243-254.

Mattson P, Kowal J. The ultrastructure of functional mouse adrenal cortical tumor cells in vitro.

Differentiation 18 Aug. 1978; 11(2): 75-88.

Nevalainen TJ. Ultrastructure of gastric leiomyosarcoma. Virchows Arch A Pathol Anat Histol.

4 Aug. 1978; 379(1): 25-33.

Timme AH. Hypersplastic foci in precancerous rat liver : light microscopic and electron microscopic study. J Natl Cancer Inst. Aug. 1978; 61(2): 407-409.

Nishi M. A cell line derived from BBN (N-butyl-N-[4-hydroxybutyl]- nitrosamine) induced rat bladder cancer : establishment and scanning electron microscopic cell surface characteristics.

Acta Med Okayama. Jul. 1978; 32(3): 181-205.

Machida S, et al. Establishment of a human rectal cancer cell line producing carcinoembryonic antigen. Gann. Dec. 1977; 68(6): 775-780.

Rodrigues MM, Furgiuele FP, Weinreb S. Malignant fibrous histiocytoma of the orbit. Arch Opththalmol. Nov. 1977; 95(11): 2025-2028.

Kovacs K, et al. Spontaneous pituitary adenomas in aging rats: a light microscopic, immunocytological and fine structural study. Beitr Pathol. Sep. 1977; 161(1): 1-16.

Machinami R, Morita T. Immunoelectron microscopy of surface immunoglobulins in pseudolymphoma of the lung : report of a case. Acta Pathol Jpn. Jul. 1977; 27(4): 547-556.

Kalderon AE, et al. Ultrastructure of myeloma cells in a case with crystalcryoglobulinemia.

Cancer. Apr. 1977; 39(4): 1475-1481.

Valenta LJ, Michel Bechet M. Electron microscopy of clear cell thyroid carcinoma. Arch Pathol Lab Med. Mar. 1977; 101(3): 140-144.

Chedid A, Bundeally AE, Mendenhall CL. Inhibition of hepatocarcinogenesis by adrenocorticotropin in aflatoxin B1-treated rats. J Natl Cancer Inst. Feb. 1977; 58(2): 339-349.

Delaney WE. Transplantable murine salivary gland carcinoma (myoepithelioma) : I. Biologic behaviour and ultrastructural features. J Natl Cancer Inst. Jan. 1977; 58(1): 61-65.

Bloustein PA, Silverberg SG, Waddell WR. Epithelioid sarcoma: case report with ultrastructural review, histogenetic discussion, and chemotherapeutic data. Cancer Dec. 1976; 38(6): 2390-2400.

Alvira MM, Mandybur TK, Menefee MG. Light microscopic and ultrastructural observations of a metastasising malignant epithelioid schwannoma. Cancer. Nov. 1976; 38(5): 1977-1982.

Pushkina IP, Krechetova GD, Shapot VS. Correlation of membrane bound and free ribosomes in normal rat liver, Zajdela hepatoma rat liver and ascite cells proper. Biokhimiia Nov. 1976; 41(11): 1940-1944.

Yasuzumi G, et al. Effects of bleomycin on human tongue carcinoma cells as revealed by electron microscopy. Cancer Res. Oct. 1976; 36(10): 3574-3583.

Parmley RT, et al. Hodgkin’s disease and myelomonocytic leukemia : an ultrastructural and immunocytochemical study. Cancer. Sep. 1976; 38(3): 1188-1198.

Toth B, Malick L, Shimizu H. Production of intestinal and other tumors by 1, 2dimethylhydrazine dihydrochloride in mice : I. A light and transmission electron microscopic study of colonic neoplasms. Am J Pathol. Jul. 1976; 84(1): 69-86.

Ingleton PM, Hancock MP. Pituitary growth hormone and somatotrophs in rats bearing chemically induced hepatomas. Virchows Arch B Cell Pathol. 29 Apr. 1976; 20(3): 253-260.

Rorat E, Fenoglio C. The ultrastructure of a poorly differentiated adenocarcinoma of the human tuba uterinae. Oncology. 1976; 33(4): 167-169.

Lupulescu A, Pinkus H. Electron microscopic observations on rat epidermis during experimental carcinogenesis. Oncology. 1976; 33(1): 24-28.

Glaso M, Iversen OH, Hovig T. The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes: a morphometric study of so-called “dark cells” and their putative role in epidermal carcinogenesis. Virchows Arch B Cell Pathol Incl Mol Pathol. 1989; 56(4): 221-235.

Leong AS, Chawla JC, Teh EC. Pituitary thyrotropic tumour secondary to long standing primary hypothyroidism. Pathol Eur. 1976; 11(1); 49-55.

Laitio MT, Nevalainen TJ. Ultrastructure of primary lymphosarcoma of testis. Z Krebsforsch

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203 204 205 206

Klin Onkol Cancer Res Clin Oncol. 27 Oct. 1975; 84(2): 189-192.

Katenkamp D, Stiller D. Cellular composition of the so called dermatofibroma (histiocytoma cutis). Virchows Arch A Pathol Anat Histol. 18 Sep. 1975; 367(4): 325-336.

Hirano A, Matsui T, Zimmerman HM. The fine structure of ependymoma. No Shinkei Geka Jul. 1975; 3(7): 557-563.

Schneider P. Malignant lymphoma resembling Burkittâ&#x20AC;&#x2122;s tumour in rhesus monkeys: (light and electron microscopic studies). Beitr Pathol. Jul. 1975; 155(3): 285-296.

Chernina LA, Shvemberger IN. Ultrastructure of the cellular elements of mouse rhabdomyosarcoma. Vopr Onkol. 1975; 21(11): 65-75.

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CUADROS DE RIBOSOMAS LIBRES Cuadro – I (Numerous free ribosomes) Nº de Caso

Diagnóstico/ Información

1 9 10

Carcinoma papilar invasivo Mammary Paget disease Tumor Intestinal

24

Neuroblastoma

26 30 31

Small cell carcinoma of the ovary Malignant fibrous histiocytoma Adenocarcinoma of ovary clear cell

38 44

Hepatocarcinogenesis induced by ethionine Pituitary fibrosarcoma

48

Rat malignant fibrous histiocytoma

50 52

Small cell osteosarcoma Mouse mammary gland adenocarcinoma

59 75

Human osteosarcoma Testicular seminoma

87 88

Phylloides tumor of the prostate Malignant Choroid Plexus Papiloma

91 105 107 108

Malignant fibrous histiocytoma Small cell carcinoma of ovary Mammary Paget disease Adenocarcinoma of stomach

117

Small cell carcinoma of the ovary

118 123 125 127 129

Neuroblastoma Rat Hepatocarcinogenesis induced by ethionine Small cell osteosarcoma Transplantable Rat malignant fibrous histiocytoma Pituitary adenoma

131 132

Large malignant choroid plexus Control neurocitoma

142 151 153

Testicular seminoma Mouse mamary gland adenocarcinoma Osteosarcoma

163 178 191 193

Mt TW15 mamosomatrofic tumor Hyperplasic foci in precancerous rat liver Human tongue carcinoma cells Intestinal tumors

206

Nasopharyngeal carcinoma

Amostra – 36 Fenótipos de malignidade – 33 92% Tumores tejidos malignos

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Cuadro – II (rich in free ribosomes) Nº de Caso 8 96 106 140 197

Diagnóstico/ Informacion Oligodendroglioma Adenocarcinoma of the stomach Oligodendroglioma Naked nuclei in breast aspirate smears Epidermal carcinogenesis

Amostra – 5 Fenótipos de malignidade – 5 100% Tumores of tejidos malignos

136

RIBOGRAMA PROJECT Cuadro – III (Abundant free ribosomes) NºCaso 3 27 28 29 41 45 51 69 71 80 90 95 98 104 115 116 128 138 145 146 156 160 165 166 168 171 173 176 183 187 188 189 199 201

Diagnóstico/ Informacion Adrenocortical carcinoma Carcinoma of the endometrium Adrenocortical oncocytoma Chemical induced mouse hepatoblastoma Carcinoma do endometrium Epithelial malignant schwannoma cell line Ewing’s sarcoma Brain tumor in doges Vaginal hemangiopericytoma Mouse hepatoblastoma Chemically induced mouse hepatoblastoma Adrenocortical carcinoma Carcinoma of thyroid gland Ductal papillary adenocarcinoma Carcinoma of endometrium Adrenocortical oncocytoma Epithelioid malignant schwannoma cell lise Mouse hepatoblastoma Vaginal hemangiopericytoma Brain tumor Extra skeletal Ewing’s sarcoma Neuroblastoma Epithelial tumors induced in rat kidney Hepatoblastoma of foetal liver Hepatocellular tumors Stromal sarcoma of breast Cholangiocarcinoma Adrenal cortical tumour Pseudolymphoma of the lung Salivary gland carcinoma Epithelioid sarcoma Malignant epithelioid schwannoma Primary lymphosarcoma of testis Ependymoma

Amostra – 36 Fenótipos de malignidade – 34 94,4% de Tumores o tejidos malignos

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Cuadro – IV (many free ribosomes) Nº de Caso 33 37 49 60 73 89 101 102 112 122 124 126 130 137 143 155 164 196 202

Diagnóstico/ Informacion NCI-H295 R cells Pulmonary clear cell carcinoma Colorectal epithelium tumors Subependymal giant cell tumor Pancreatic islet cell tumor Neuroblastoma cell line Primary osteoblast like cells Human pituitary tumor NCI-H 295 R cells (human adrenal cell lines) Pluripotent human germ cell tumor derivated cell line Pulmonary clear all carcinoma Colorectal tumors Human neuroblastoma cell line Malignant fibrous histiocytoma Pancreatic islet cell tumor Malignant lymphoma Spontaneous testicular neoplasm Epidermis experimental carcinogenesis Malignant lymphoma

Amostra – 19 Fenótipos de malignidade – 16 84,2% de tumores o tejidos malignos

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Cuadro – V (Increased number of free ribosomes) Nº de Caso 6 11 15 19 22 23 32 34 35 47 62 77 79 84 100 109 110 113 120 133 141 157 159 167 181 185 186 204

Diagnóstico/ Informacion Human erythroleukemia cells Rat liver hepatocarcinoma induced Ewing’s sarcoma Nasopharyngeal carcinoma Adrenocortical carcinoma Thalamic tumor Pseudoductular changes in pancreas pigs Leiomyoblastoma of the uterus Liver tumor rats Human erythroleukemia cells Papillary thyroid carcinoma Medullary carcinoma of the thyroid Cutaneous neurofibromas R and T head and neck carcinomas Human erythroleukemia cells Transplantable hepatomas Adrenocortical carcinoma Pseudo-ductular changes in pancreas of pigs Embryonal Rhabdomyosarcoma Seminoma and parathyroid adenoma Medullary carcinoma Oesophageal epithelium dysplastic foci of the thyroid Pituitary adenomas Uveal malignant melanoma Malignant fibrous histiocytoma of the orbit Clear cell thyroid carcinoma Hepatocarcinoma Carcinogenesis induced by 4- dimethylaminobenzene

Amostra – 28 Fenótipos de malignidade – 22 78,6% de Tumores o tejidos malignos

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Cuadro – VI (referencia a “free ribosomes”) Nº de Caso 2 5 7 12 14 16 17 21 25 36 39 40 42 43 46 55 56 57 63 64 65 66 67 68 70 72 74 76 78 81 82 83 85 86 92 94 97 99 103 111 119 120 121

Diagnóstico/ Informacion Glial C6 cells transformed Administration of ethionine female rats Bronchogenic carcinoma Neuroblastoma cell lines Malignant extra renal rhabdoid tumor Colorectal polyps Hepatoma transplantable Ewing’s sarcoma Carcinoma of thyroid gland Cortical dendritic spines Human germ cell tumor derived cell line Embryonal rhabdomyosarcoma Tapioca melanoma of the iris “Lipomatous” lesions in the liver of B6 C3 F1 mice Rat bladder tumor cell lines Poorly differentiated rhabdomyosarcoma Lymphocytic leukaemia tumor Adenocarcinoma from apocrine glands in dogs Carcinoma of lung Friend erythroleukemia cell growth Hodgkin’s disease Hepatoma cells Hepatoma cells Astroblastoma Endometrial adenocarcinoma Clear odontogenic tumor Cell condrosarcoma Sphenoidol ridge meningioma Osteosarcoma cell line Malignant fibrous histiocytoma Rat pheochromocytoma cell Carcinoma of endometrium Myelogenous leukaemia cell line Seminoma and parathyroid adenoma Myeloma cells Congenital fibrosarcoma Human tumoral ependymal cell lines Ductal adenocarcinoma of the pancreas Carcinoma rat testis Human adrenal cell lines Cerebral small cell tumor Embryonal rhabdomyosarcoma Nevic corpuscle

140

RIBOGRAMA PROJECT 134 135 136 139 144 148 149 150 154 158 161 162 169 170 174 175 177 180 182 184 195 198 200 205 208

Myelogenous leukaemia cell line Carcinoma of the endometrium Adrenal chromaffin and pheochromocytoma N-methyl-N-amilnitrosamina induced rat oesophageal carcinogenesis Clear odontogenic tumour Rat ascitis hepatoma cells Rat ascitis hepatoma cells Carcinoma of the lung Adenocarcinomas (in dogs), aprocrine glands Branchiometric paragangliomes Adrenocortical carcinomas Melanocytomas of the optic nerve Transitional cloagenic carcinomas Maxilofacial synovial sarcoma Cultured hepatoma cells Monoclonal Hodgkin cells cultured Gastric leiomyosarcoma Establishment of a human rectal cancer cell line Pituitary adenomas Myeloma cells Adenocarcinoma of the human tuba uterinae Pituitary thyrotrophic tumor Dermatofibroma (histiocytoma) Friend erythroleukemia cell growth Human neuroblastoma cell

Amostra - 70 FenĂłtipos de malignidade â&#x20AC;&#x201C; 59 84,29% de Tumores o tejidos malignos

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XXI - METFORMIN PREVENTS CARCINOGEN - INDUCED TUMORIGENESIS (METFORMIN PREVENTS CANCER) A. Ferreira-Alemao, MD PhD UNIVERSIDAD COMPLUTENSE DE MADRID

Metformin, as anti-diabetic drug, is one of the FDA-approved drugs with a near-perfect safety record, low cost, and favorable side-effect profile. Between 1990 and 2011 alone, (1) over 1,000 published studies have yielded confirmatory data on its numerous anti-aging properties, from weight loss and glucose control to cardiovascular disease and cancer defense. As this medical body of evidence grows, ongoing research validates powerfully this idea. In one of the largest studies of its kind, it was analyzed the cancer risk among 8,000 diabetics treated with metformin. (2) Over a 10-year period, it was observed a 54% lower incidence of all cancers compared to the general population.

Metformin not only exerted a major protective effect against cancer development, but those who developed cancer exhibited a significantly higher survival rate, including those with malignant cancers of the lung, colon, and breast. Of equal significance was the finding that the earlier the metformin regimen was initiated, the greater the preventive benefit. Given that diabetics are predisposed to a wide array of cancers — of the breast, colon, liver, pancreas, kidney, endometrium, among others (3-5) — these results have profound implications for all population.

In this review, the most recent data supporting metformin’s anti-cancer mechanisms are detailed. You will learn of its specific mechanisms of action, which shed further light on the link between obesity, diabetes, and cancer initiation. Through this review you will also discover how metformin induces cancer cell death at their earliest stages of development via metabolic pathways, which also promote weight loss and optimal glucose control. Metformin slashes cancer risk in multiple clinical trials. This is of great medical importance and can be analysed under cancer epidemiology aspect. (6) Duncan observed that this was the most compelling amongst a rapidly growing set of studies, all suggesting that metformin might induce profound effects in preventing a wide range of cancers while improving prognosis in people who do develop malignancies.(6) Libby et al.(8), in this issue of Diabetes Care, report a lower incidence of a broad range of cancers among diabetic patients treated with metformin. Specifically, by linking data from a population-based diabetes registry with those from a drug use registry and a cancer registry, they effectively followed medication use in over 8,000 diabetic individuals and ascertained over 700 incident cancers. They found, over a 10-year period, that metformin use was associated with a 54% (95% CI 47–60) lower crude and a 37% (25–47) lower adjusted incidence of cancer. From the survival analysis they performed adjusting for age, sex, BMI, smoking status, and a social deprivation index, one can estimate the association in absolute terms: considering the cancer rate of 11.6% for non–metformin users, for every 23 patients receiving metformin in this cohort one fewer developed cancer (number needed to treat = 1/[0.37 × 0.116]). Their findings are internally consistent. In patients with the longest history of metformin use prior to cancer onset, incidence tended to be lowest.

Metformin users who developed cancer also had a greater survival postdiagnosis. A protective association of similar degree was documented against lung, bowel, and breast cancer.

In a cohort study of more than 12,000 patients, metformin users died of cancer 30% less often than those taking another category of drug called sulfonylureas .(6,7) Of equal and even greater significance, people taking insulin had a 90% greater death rate than the metformin users in that study.(6) 142

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In other study of different design, people taking metformin for diabetes control for more than 36 months had a 72% lower risk of developing cancer than those on other regimens.(6,9) Similarly, in a third study, metformin users had a 62% lower risk of developing cancer, compared with those who had never used metformin.6,10 Of significance, that study also showed an increased risk of cancer in people who were taking insulin or oral antidiabetes drugs other than metformin.

Thereâ&#x20AC;&#x2122;s additional evidence that metformin not only prevents cancer from developing, but also helps to improve the prognosis in patients who do develop tumors. In one study of breast cancer patients on chemotherapy, 24% of those who were also taking metformin had a complete response rate, compared with just 8% for those not taking it.(6,11)As a result of these "incidental" findings, scientists have initiated several clinical trials to examine the impact of metformin as formal additional treatment for breast and other cancers.(6,12) Numerous recent studies further support a close association between metformin use and substantially reduced cancer incidence, along with improved survival.(13-17) These observations raise the question how a diabetes drug protect against certain cancers. Obesity and diabetes are a direct causative factor in the development of a wide range of cancers. Diabetics have as much as a 41% increased risk for virtually all cancer types compared to healthy people. Elevated blood sugar alone increases the risk of certain cancers, including those of the kidney, pancreas, and skin (melanoma).(16,18,) Obesity increases cancer risk for more than a dozen different cancers.(6) A 59%increase in cancer risk has been documented for every 5-unit increase in body mass index (BMI) alone.(6,21) Studies show that obesity is responsible for up to 20% of cancer deaths in women.(21) The link between diabetes and obesity and cancer points to the underlying mechanisms of action by which metformin works as a cancer-preventing agent. Metformin operates at the molecular level by activating adenosine monophosphate-activated protein kinase or AMPK, a molecule essential to life. AMPK or its molecular analogs are present in virtually all living organisms.(6,22) It also happens to be intimately involved in cellular processes whose dysregulation play a central role in both diabetes and obesity and cancer initiation. Diabetes and obesity result from various metabolic derangements. Cancer results from disordered regulation of cell growth. AMPK is critical to normal regulation of both metabolism and cell growth, as a result of millions of years of evolutionary development.(6) As a fuel-sensor and metabolic master switch, AMPK recognizes and responds to changes in cellular energy levels, determining how fats and carbohydrates will be used in storing or utilizing energy.(6) In metabolic terms, AMPK tells cells to conserve and generate new energy stores. In so doing, it lowers sugar output from the liver, increases glucose uptake from the blood, maintains insulin sensitivity, and ultimately lowers blood sugar.(6,22) AMPK exerts similar effects in terms of regulating cell growth and replication, instructing cells to conserve energy, slowing and often shutting down aberrant cell growth entirely. In essence, when AMPK is activated, incipient cancer cells starve themselves to death for lack of adequate energy supplies.(23) We can naturally activate AMPK in our bodies through several time-honored mechanisms. Calorie restriction lowers cellular energy stores and activates AMPK.(24) Known to increase life span in virtually all species, calorie restriction has been shown to reduce cancer incidence and death in primate studies.(25,26 )And a recent study showed that gastric bypass surgery not only produced sustained weight loss, but also reduced cancer incidence by 42% in women patients (no effect was seen in men).(6,27) Exercise is another strong natural activator of AMPK, and studies show that people with the highest levels of physical activity are protected against cancers of the lung and colon by as much as 30%.(6,28,29) Exercise and weight loss are lifestyle changes that most of us need to make, while bariatric surgery and massive calorie restriction have more limited appeal and application as means of activating AMPK and lowering cancer risk. Metformin, a natural product of the French lilac,(30) is a safe, readily available, and inexpensive way to activate AMPK and starve cancer cells of their energy supplies.(6,31,32) In doing so, 143

RIBOGRAMA PROJECT metformin powerfully restores healthy regulation—both of metabolic factors and of those that regulate cell growth. Let’s now examine how metformin halts incipient cancers by quelling abnormal cellular proliferation, one of the earliest steps in cancer development.

Lab Studies -- Anti-Cancer Power Healthy, normal people develop incipient cancer cells in their bodies daily; these cells are normally destroyed by a number of natural processes. When those processes break down, the cancer cells are free to proliferate and form a tumor. An ideal anti-cancer drug, then, would eliminate these altered, "precancerous" cells before they could replicate and become invasive and malignant.(33) Even in their earliest stages, aggressive cancer cells are notoriously energy-hungry, burning calories at a frenetic rate as they grow out of control.(34) For that reason, targeting cancer cell metabolism now stands at the forefront of cancer prevention research.34 With its potent ability to shut off the cellular energy pipeline by activating AMPK, metformin is showing its value in preventing or slowing a host of cancer types in laboratory studies.

The consequences of AMPK activation by metformin are numerous. Metformin, added to cultures of many different cancer cell types, blocks proliferation by "stalling" cells at one of several phases of the cell replication cycle, preventing them from reproducing. (35-38) Metformin’s ability to starve cancer cells of energy also enhances the rate of cell death by the process known as apoptosis, one of the body’s natural means of cancer control.(35,39) Perhaps the most detailed picture of metformin’s antiproliferative actions comes from a 2011 study in France.(39) Researchers there added metformin to melanoma skin cancer cells in culture, and monitored the effects. At 24 hours, metformin had starved the cancer cells to the point that their replicative cell cycle was arrested. By 72 hours, the cells underwent autophagy, a mechanism whereby starving cells literally "eat themselves" in a desperate attempt to survive. And by 96 hours, the cancer cells began dying off en masse by apoptosis.

Several additional antiproliferative mechanisms have recently been demonstrated for metformin in addition to its effects on the AMPK energy-sensing pathway.(36,40-43) That ability to act by multiple mechanisms is called pleiotropy. It is powerfully beneficial because it prevents development of resistance to any one pathway that is seen much more commonly with natural products such as metformin than with mono-targeted pharmaceutical drugs. The combined effect of all of metformin’s pleiotropic mechanisms is a marked reduction of tumor growth in lab animals implanted with human cancer cells.(47) Metformin Prevents Cancers in Non-Diabetic Individuals Perhaps the most exciting news to come out of the recent surge in interest in metformin is that the drug can prevent cancers from forming in animals and humans who are not diabetic. As a "mimicker" of a calorierestricted state, that might be expected of metformin, given that calorie restriction is such a potent cancerpreventive strategy.(34,50-52) Since 2008, a small explosion of studies has appeared demonstrating how effective metformin can be in this context, ultimately suggesting that it should be taken regularly by anyone who wants to reduce their risk of dying from cancer. Research now demonstrates that metformin, provided orally to lab animals, prevents deadly colorectal cancers(53) (the second leading cause of cancer deaths in the US, and an astonishingly preventable disease(54)). Metformin suppresses intestinal polyp growth, a precursor of colorectal cancer, in mice predisposed to that disease.(55 ). And, in a study of chemically induced colon cancer, metformin significantly reduced formation of so-called "aberrant crypt foci," which in humans represent an early stage in cancer development.(56) Those studies led to the first human study of metformin as a cancer preventive agent in non-diabetic people.

Researchers studied 26 non-diabetic people with aberrant crypt foci that had been found on routine 144

RIBOGRAMA PROJECT colonoscopy.57They randomly assigned them to receive metformin 250 mg per day, or no treatment, and then performed repeat colonoscopy one month later. The metformin group had a significant decrease in the number of aberrant crypt foci, from nearly 9 per patient down to about 5 per patient, while control patients had no change. This represent a 55% reduction in this cancer precursor in patients taking low-dose metformin.

Chemoprevention studies now also demonstrate similar effects in other cancers. Mice supplemented with oral metformin, exposed to a potent tobacco carcinogen, developed 53% fewer lung cancers than did control animals.58And when metformin was administered by injection, that protection rate rose to 72%.

Breast cancer prevention would represent a huge forward stride in extending human life span and reducing suffering. Thereâ&#x20AC;&#x2122;s encouraging data here as well. Mice given metformin in their drinking water for 13 days prior to injection with a powerful breast carcinogen had significantly delayed onset of tumor development.(59) Several other studies have demonstrated that metformin-supplemented mice experience a reduction in proliferation of cancer-prone breast cells and inhibition of tumor (32) There is now a tremendous body of literature showing that metformin prevents cancer cells from proliferating, and moreover it prevents clinically relevant human cancers from developing, even in non-diabetic, non-obese individuals(60)As a result, one might expect to see large clinical trials of metformin in healthy older adults as a cancer chemopreventive agent. Sadly, even though calls for such studies are gathering strength, to date no trial has been designed, let alone implemented.(61-63) Given metforminâ&#x20AC;&#x2122;s impressive safety record over nearly 50 years of clinical use,(44) there is simply no reason for sensible people to wait for an "official" medical establishment recommendation. People who are concerned about their growing risk of cancer should simply speak to their physicians now, and present them with a synopsis of the data, so that they can begin potentially lifesaving use of metformin today.

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References 1. Andújar-Plata P, Pi-Sunyer X, Laferrère B. Metformin effects revisited. Diabetes Res Clin Pract. 2011 Oct 13.

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30. Graham GG, Punt J, Arora M, et al. Clinical pharmacokinetics of metformin. Clin Pharmacokinet. 2011 Feb 1;50(2):81-98.

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38. Rattan R, Giri S, Hartmann LC, Shridhar V. Metformin attenuates ovarian cancer cell growth in an AMP-kinase dispensable manner. J Cell Mol Med. 2011 Jan;15(1):166-78.

39. 39 Tomic T, Botton T, Cerezo M, et al. Metformin inhibits melanoma development through autophagy and apoptosis mechanisms. Cell Death Dis. 2011;2:e199.

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42. Micic D, Cvijovic G, Trajkovic V, Duntas LH, Polovina S. Metformin: its emerging role in oncology. Hormones (Athens). 2011 Jan-Mar;10(1):5-15.

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45. Gotlieb WH, Saumet J, Beauchamp MC, et al. In vitro metformin anti-neoplastic activity in epithelial ovarian cancer.Gynecol Oncol. 2008 Aug;110(2):246-50.

46. Zakikhani M, Dowling RJ, Sonenberg N, Pollak MN. The effects of adiponectin and metformin on prostate and colon neoplasia involve activation of AMP-activated protein kinase. Cancer Prev Res (Phila). 2008 Oct;1(5):369-75.

47. Liu B, Fan Z, Edgerton SM, Yang X, Lind SE, Thor AD. Potent anti-proliferative effects of metformin on trastuzumab-resistant breast cancer cells via inhibition of erbB2/IGF-1 receptor interactions. Cell Cycle. 2011 Sep 1;10(17):2959-66.

48. Antonoff MB, Dâ&#x20AC;&#x2122;Cunha J. Teaching an old drug new tricks: metformin as a targeted therapy for lung cancer. Semin Thorac Cardiovasc Surg. 2010 Autumn;22(3):195-6.

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XXII - SIMVASTATIN INHIBITS CANCER CELL GROWTH A. Ferreira-Alemao, MD PhD UNIVERSIDAD COMPLUTENSE DE MADRID

Administration of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, to ambulatory patients is associated with a lower incidence of long-term adverse cardiovascular events, including death, myocardial infarction, stroke, atrial fibrillation, and renal dysfunction. However, increasing clinical evidence suggests that statins, independent of their effects on serum cholesterol levels, may also play a potential role in the prevention and treatment of cancer. Specifically, statins have been shown to exert several beneficial antineo-plastic properties, including decreased tumor growth, angiogenesis, and metastasis. The feasibility and efficacy of statins for the prevention and treatment of cancer is reviewed.

The statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) have been proven to be effective in lowering cholesterol and as anti-lipid agents against cardiovascular disease. Recent reports demonstrate an anticancer effect induced by the statins through inhibition of cell proliferation. Probably, these effects are due to suppression of the mevalonate pathway leading to the depletion of various downstream products that play an essential role in cell cycle progression, cell signaling and membrane integrity. To date, although many hypotheses have been proposed, the exact mechanism at the basis of cancer cell growth arrest induced by statins is not known. In this study, we have demonstrated that Simvastatin, at a dose of 20 µM for 24-72 h, induced in cancer cells but not in normal cells precise features of apoptosis including increased DNA fragmentation while, at the molecular level Simvastatin induced overexpression of the pro-apoptotic gene Bax together with an inhibition of BCL-2, the gene that has the well-known function of protecting cells from apoptosis. The Simvastatin-mediated induction of apoptosis in similar cancer cells but not in normal cells is very interesting and may be at the basis of cancer therapy using statins, usually in combination with chemotherapy or to be used as a cancer protective drug. Simvastatin may, thus, play a dual prophylactic role as a lipid-lowering drug for the prevention of heart disease and as an anticancer agent to prevent certain types of cancers.

INTRODUCTION 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, are commonly-used drugs for the treatment of hypercholesterolemia (1, 2). Statins decrease low-density lipoprotein (LDL) cholesterol levels by inhibiting HMG-CoA reductase. HMG-CoA reductase in turn catalyzes the conversion of HMG-CoA into mevalonate and is the rate-limiting step in hepatic cholesterol biosynthesis (3). Clinically, statin treatment is associated with a reduction in atherosclerotic plaque formation and a stabilization of pre-existing “vulnerable” atherosclerotic plaques (4). Moreover, statins have been shown to decrease the incidence of adverse cardiovascular outcomes, including death, myocardial infarction, stroke, atrial fibrillation, and renal dysfunction in ambulatory patient populations (2, 5-13). Statin administration is also associated with a lower incidence of adverse cardiovascular outcomes after invasive procedures such as percutaneous transluminal coronary angioplasty (9) and cardiac (14,15), vascular (16,17) and noncardiovascular (19) surgery. However, the beneficial effects of statin therapy are not limited to patients with hypercholesterolemia. Several randomized clinical trials have shown that, even in patients with normal total and LDL cholesterol levels, statin administration is associated with less cardiovascular morbidity and mortality (7,8). Statins thus exert pleiotropic effects independent of their effects on cholesterol (20).

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RIBOGRAMA PROJECT Although the exact mechanisms by which statins reduce the likelihood of cardiovascular events have yet to be fully elucidated, the metabolite of HMG-CoA reductase, mevalonic acid, is a precursor of cholesterol and the isoprenoid intermediates farnesyl and geranyl-geranyl pyrophosphate. These intermediates are essential for the post-translational modification of intracellular G-proteins, such as Rho, Rac, and Ras, that regulate endothelial, platelet, and leukocyte function (21,23). Statins have also been shown to modulate vascular remodeling by inhibiting cellular matrix metalloproteinases and transcription factors, such as nuclear factor-ÎşB (23).

In patients with acute coronary syndromes or idiopathic dilated cardiomyopathy, statin therapy has been shown to reduce untoward inflammatory activity, including changes in C-reactive protein (CRP), serum amyloid A, tumor necrosis factor alpha (TNF-Îą), interleukin-6, and brain natriuretic peptide levels (21,24,25). Moreover, statins have been reported to decrease serum levels of the inflammatory marker CRP within 14 days of administration, suggesting an acute protective role for these drugs (26). Statins have also been shown to reduce tissue injury in models of ischemia and reperfusion in several organs, including the heart, lung, brain, kidney, and gut (19,27-30). Further, statins have been shown to attenuate vasoconstriction by increasing endothelial nitric oxide (NO) activity, a benefit seen within 6 weeks of the start of treatment (31). Statins thus exert pleiotropic effects, independent of cholesterol reduction, that have direct antiatherosclerotic, antithrombotic, and anti-inflammatory impacts (23,32-34).

Increasing evidence suggests that statins might be useful for cancer prevention and/or treatment through their interactions with essential cellular functions, such as cell proliferation and differentiation (35,36). For example, both in vitro and in vivo studies have demonstrated that statins inhibit tumor growth and induce apoptosis in a variety of tumor cells, including melanoma (37), glioma (38), neuroblastoma (39), and leukemia cell lines (40).

Additionally, several clinical trials have also assessed the antitumor activity of statins (41- 46). The potential role of statins in both cancer prevention and treatment is reviewed.

ANTITUMOR EFFECTS OF STATINS Inhibition of Tumor Cell Growth Cholesterol is a major structural component of cell membranes, and the cholesterol biosynthetic pathway is closely related to cell-growth processes. Statins reduce not only serum cholesterol levels but also mevalonate synthesis by inhibiting HMG-CoA reductase. Mevalonate is a precursor of several major products regulating the cell cycle, including dolichol, geranylpyrophosphate (GPP) and farnesyl-pyrophosphate (FPP) (3). Dolichol has a stimulatory effect on DNA synthesis and is linked to several tumor cell proteins (47). GPP and FPP cause isoprenylation of the intra-cellular G-proteins Ras and Rho, which in turn regulate the signal transduction of several membrane receptors crucial for the transcription of genes involved in cell proliferation, differentiation, and apoptosis.

Ras and Rho gene mutations are found in a variety of pancreas (90%), colon (50%), lung (30%), thyroid (50%), and myeloid leukemia (30%) tumor types (3-6). Statins inhibit dolichol, GPP and FPP production, and block tumor cell growth in vitro and in vivo (48). For example, lovastatin has been shown to stabilize the cell cycle kinase inhibitors p21 and p27, and to arrest breast cancer cell lines in the G 1 phase of the cell cycle (49).

Similarly, cerivastatin has been demonstrated to inhibit Ras- and Rho-mediated cell proliferation (50). These observations have led several investigators to hypothesize that statins might inhibit the growth of a variety of tumor cell types, including prostate, gastric, and pancreatic carcinoma, as well as colon adenocarcinoma, neuroblastoma, glioblastoma, mesothelioma, melanoma, and acute myeloid leukemia cells (42, 51-56).

Interestingly, statins also modify normal endothelial, fibroblast, and smooth muscle cell growth (57,58).

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RIBOGRAMA PROJECT However, normal cells appear to be more resistant to the antiproliferative effects of statins relative to tumor cells, which are much more likely to proliferate (59,60).

Inhibition of Angiogenesis Angiogenesis plays an important role in primary tumor growth and metastasis (61). Statins have been reported to both stimulate (62-64) and inhibit (65,66) blood vessel formation depending upon the tumor cell type (67).

For example, high-dose cerivastatin decreased tumor vascularization by 51% in a murine Lewis lung cancer model (68). Statins have been shown to decrease vascular endothelial growth factor production (69) and to inhibit capillary tube formation (66,70). In contrast, statins have also been shown to stimulate protein kinase B, which in turn activates endothelial nitric oxide synthase (eNOS) and increases proangiogenic activity (64). This NO-mediated effect depends on caveolin, a protein that downregulates eNOS activity (71). Endothelial cells with low caveolin expression seem to be extremely sensitive for statin-induced angiogenesis. Finally, there is growing evidence that the effects of statins on angiogenesis are concentration dependent. Weis et al. showed that low concentrations (0.5 mg/kg per day) of cerivastatin and atorvastatin enhanced endothelial cell proliferation, whereas high concentrations (2.5 mg/kg per day) significantly inhibited angiogenesis (68).

Induction of Apoptosis Several experimental cancer models have shown that statins exert proapoptotic properties in a variety of tumor cells. For example, lovastatin induces a profound apoptotic response in cells derived from juvenile monomyelocytic leukemia, pediatric solid malignancies (e.g., rhabdomyosarcoma and medulloblastoma), malignant mesothelioma, astrocytoma, and squamous cell carcinoma of the cervix, head, and neck (53,59,72,73). Further, this statin-mediated apoptotic effect is also seen in cell lines treated with cerivastatin (74,75). In fact, Wong et al. found that cerivastatin is 10 times more potent in inducing apoptosis in acute myeloblastic leukemia (AML) cell lines than other statins (75). Additionally, tumor cells themselves differ significantly in their sensitivity to statin-induced cell death. AML cells (76) and neuroblastoma cells (77) seem to be particularly sensitive to statin-induced apoptosis, whereas acute lymphoblastic leukemia cells are relatively insensitive.

Proposed mechanisms for statin-mediated apoptosis include an upregulation of proapoptotic protein expression (e.g., Bax, Bim) (78) combined with decreased anti-apoptotic protein expression (e.g., Bcl-2) (40).

For example, lovastatin increases Bim protein levels and induces cell death in human glioblastoma cell lines (79). These effects have been seen in both solid tumor and hematologic malignancies. Statins have also been shown to activate caspase proteases involved in programmed cell death. Cafforio et al. showed that cerivastatin caused cell death in human myeloma tumor cells by activating caspase-3, caspase-8, and caspase-9 (80). Similarly, lovastatin induces apoptosis in prostatic epithelium and leukemia cells through activation of caspase-3 and caspase-7, respectively (81,82).

Repression of Tumor Metastases Several lines of evidence suggest that statins impair the metastatic potential of tumor cells by inhibiting cell migration, attachment to the extracellular matrix, and invasion of the basement membrane. In addition to reducing endothelial leukocyte adhesion molecule (e.g., E-selectin) (83) and matrix metalloproteinase (MMP)-9 expression (84), statins have been shown to inhibit epithelial growth factorâ&#x20AC;&#x201C;induced tumor cell invasion (60).

Both in vitro and in vivo studies have shown that fluvastatin and lovastatin reduce liver tumor formation and growth of established liver metastases in pancreatic cancer cells (85). Similarly, atorvastatin has been shown to decrease melanoma cell metastasis (86). Statins have also been shown to suppress lung metastasis of 152

RIBOGRAMA PROJECT renal and mammary carcinoma cells (87,88), as well as liver metastasis of colon adenocarcinoma cells (85,89). However, not all studies have confirmed that statins reduce tumor metastases. For example, lovastatin failed to inhibit colon carcinoma and glioblastoma cell migration and invasion (89).

STATINS AND CANCER RISK/PREVENTION Experimental data suggesting a potential carcinogenicity and mutagenicity of statins date back to the early 1990s. In vitro experiments of epithelial and fibroblastic cell lines showed that lovastatin induced prometaphase retardation and chromosome lagging during cellular meta-phase and anaphase (90). This led some to speculate that statins might interfere with centromere development and function, causing a mitotic disorder. Supporting this notion were studies demonstrating that simvastatin administered at a dose approximately 250 times higher than the dose normally used to treat hypercholesterolemia caused thyroid hypertrophy and follicular cell adenomas in rat strains (91). Similarly, lovastatin administration at high dosage levels (500 mg/kg per day) was associated with a higher incidence of hepatocellular and pulmonary cancers (92). Although fluvastatin was not found to cause liver tumors in rats and mice, it was found to be associated with a higher risk for thyroid neoplasms and forestomach papillomas in rodents (93). Interestingly, statinassociated carcinogenicity in most of these studies was limited to dosages much higher than that commonly used to treat hypercholesterolemia in humans.

In contrast, other in vitro and in vivo studies have suggested that statins might exert an anticarcinogenic effect.

For example, toxicity studies in rats, dogs, and monkeys with lovastatin at doses up to 180 mg/kg per day demonstrated no carcinogenic effects (92). Further, pravastatin and simvastatin were shown to suppress the development of 1,2-dimethylhydrazineâ&#x20AC;&#x201C;induced colon cancer in mice (94). Similarly, pravastatin has been shown to reduce the incidence and volume of N-nitrosomorpholineâ&#x20AC;&#x201C;induced hepatic neoplastic nodules and to reduce N-methyl-N-nitrosoureaâ&#x20AC;&#x201C;induced colon carcinogenesis (95,96) Further confusing the issue is the fact that human data regarding the cancer risk with statin administration are also conflicting. A cohort study of more than 12,488 men and women revealed an inverse association between cholesterol level and cancer incidence, including lung, colorectal, pancreatic, and bladder cancers, and leukemia (97). In contrast, several cholesterol-lowering trials have reported a tendency toward a higher number of cancer deaths in participants treated with cholesterol-lowering agents. The incidence of breast cancer in a prospective trial was 5.5% among pravastatin treated patients versus 0.3% in the placebo group (8). Additionally, a retrospective analysis of 2,463 women revealed a 1.5-fold higher risk for breast cancer among statin users (98). In a prospective study, conducted in elderly subjects, pravastatin was associated with a higher incidence of newly diagnosed gastrointestinal cancers, although the difference did not reach statistical significance (1). Furthermore, no difference in overall cancer incidence between the statin and placebo groups occurred in that trial. However, another study showed double cancer death rates, mainly of gastrointestinal cancers, in men with low serum cholesterol levels (99).

Several large investigations have failed to show a higher incidence of breast or gastrointestinal cancer. The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial, for the primary prevention of cardiovascular events, and the Long-term Intervention with Pravastatin in Ischemic Disease trial revealed a similar relative risk for overall cancer development in the pravastatin and placebo groups (100,101). The Heart Protection Study found an equal incidence of any cancer subtype in simvastatin- and placebo-treated patients (102). Moreover, later epidemiologic studies suggest that statins may prevent certain malignancies, including melanoma, and breast, colon, and prostate cancer (103,106). In that regard, the Air Force/Texas Coronary Atherosclerosis Prevention Study showed a 50% lower incidence of new cases of melanoma in lovastatintreated patients (7). Pederson et al. (107) reported a trend toward a lower cancer mortality rate in simvastatintreated participants of the Scandinavian Simvastatin Survival Study. During the median follow-up period of this double-blind trial (65 months), 35 patients and 33 patients died in the placebo and statin groups, respectively.

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RIBOGRAMA PROJECT No significant differences with respect to cancer incidence (relative risk, 0.88; 95% confidence interval [CI], 0.73–1.05; p = .147 and mortality (odds ratio [OR], 0.80; 95% CI, 0.60–1.08; p = .142) in this study population were seen after a 10-year follow-up period (108). Later case-controlled studies found a lower risk for breast cancer (72%) (104) uterine cancer (70%) (109) and prostate cancer (56%) (106) among statin users.

Additionally, the investigators of The Molecular Epidemiology of Colorectal Cancer Study, a case-controlled study, observed a 47% lower risk for colorectal cancer among long-term statin users (OR, 0.50; 95% CI, 0.40– 0.63) (103). Multivariate analysis further revealed that long-term statin therapy was also protective against colon cancer in high-risk patients, including those with a family history of colorectal cancer, hypercholesterolemia, ischemic heart disease, and inflammatory bowel disease, with an evaluated number needed to treat of approximately 2,400 patients. Interestingly, Graaf et al. reported a return to baseline risk for cancer development after withdrawal of statins, underpinning the potential protective effect of statins (110).

Finally, a systematic review of 10 cohort studies, two international studies, and 28 randomized trials revealed that low or reduced serum cholesterol concentrations were not associated with higher cancer mortality (111).

STATINS AND CANCER TREATMENT Increasing evidence suggests that statins might enhance the antitumor activity of various cytokines and chemotherapeutic agents. For example, the combined administration of lovastatin and TNF-α has been shown to inhibit the proliferation of murine melanoma and leukemia cell lines and to prolong the survival of tumorbearing mice (112,113). Synergistic interactions have also been reported between statins and chemotherapeutic agents such as cisplatin, 5-fluorouracil (5-FU), and doxorubicin. For example, pretreatment with lovastatin significantly increases 5-FU– and cisplatin-induced apoptosis in colon carcinoma cell lines (78).

Further, the anti-tumor effects of cisplatin have been shown to be augmented when combined with lovastatin in a murine melanoma model (114). Lovastatin has also been shown to enhance paclitaxel-induced apoptosis in human leukemia cell lines (115). Particularly interesting are the interactions between lovastatin and doxorubicin. As with other chemotherapeutics, lovastatin enhanced the antitumor activity of doxorubicin in murine melanoma, colon carcinoma, and lung carcinoma (116,118). However, in addition to increasing the antitumor effect, lovastatin treatment was also associated with a lower risk for doxorubicin-associated cardiotoxicity (116,119).

Several human clinical trials have investigated the anti-tumor effects of statins. In a controlled, randomized trial of 91 patients with unresectable hepatocellular carcinoma, patients were randomly assigned to receive either pravastatin (20 mg/day for 2 weeks followed by 40 mg/day) or placebo concomitant with 5-FU chemotherapy (41). None of the patients had to stop pravastatin as a result of toxicity. Further, the pravastatin-treated patients showed a reduction in tumor growth at 6 months and 1 year, and their median survival rate was double that of the control group. Similarly, a retrospective multivariate analysis of 349 patients with rectal cancer and neoadjuvant chemoradiation revealed that the response rate among statin users was fourfold higher than that of nonusers (120). Finally, another case report described the inhibition of leukemic blast cell proliferation by lovastatin in AML patients (45).

However, not all clinical studies have yielded promising results. Pravastatin therapy as a treatment option for advanced hepatocellular carcinoma was not associated with a significantly better overall survival rate, although statin therapy seemed to be more beneficial than other new treatment options, including octreotide and gemcitabine therapy (121). In a study using fluvastatin (8 mg/kg per day for 14 days every 4 weeks) in pediatric cancer patients, 10 of 12 patients died within 1–18 months because of their advanced disease stage, and laboratory assays demonstrated no significant changes during treatment (122). Knox et al. conducted a phase I trial of lovastatin administration (7.5 mg/kg per day for 21 consecutive days) in patients with recurrent or metastatic squamous cell carcinoma of the head and neck (43). Although no objective improvements in tumor response were observed, 23% of the patients experienced stable disease for more than 3 months.

Finally, in a study of 10 patients with chronic lymphocytic leukemia who received simvastatin (40 mg) daily for 154

RIBOGRAMA PROJECT 2 weeks, no significant clinical change in the patientsâ&#x20AC;&#x2122; status was observed while taking the medication (123).

However, 40% of patients developed progressive disease during the subsequent year following statin withdrawal.

Several trials have examined the feasibility and safety of high-dose statins in cancer patients. In a phase I trial of lovastatin (2â&#x20AC;&#x201C;45 mg/kg per day) in patients with solid tumors, no adverse side effects were seen with doses up to 25 mg/kg per day (46). At doses above this level myopathy occurred, but coadministration of ubiquinone (coenzyme Q) significantly decreased the severity of myopathy. Another phase I/II study also showed that lovastatin (20, 25, or 30 mg/kg for 7 days in cycles repeated monthly) was well tolerated in patients with malignant gliomas (44). Only two patients experienced mild arthralgias, and even in the absence of ubiquinone, no myopathies were seen. Together, these data suggest that high-dose lovastatin is well tolerated and might have modest anticancer activity, especially in the treatment of brain tumors. In contrast, the antitumor activity of high-dose lovastatin has not been shown in patients with advanced gastric adenocarcinoma (42). Sixteen patients received lovastatin (35 mg/kg per day for 7 consecutive days) with ubiquinone to prevent rhabdomyolysis. The treatment was repeated every 28 days. Two patients developed myalgia, and lovastatin administration had no anti-tumor effects in this patient population.

CONCLUSION Statins are commonly prescribed drugs for the prevention of cardiovascular disease resulting from hypercholesterolemia. However, increasing evidence suggests that statins exert pleiotropic effects, independent of cholesterol reduction. In addition to their well-described antiatherosclerotic, antithrombotic, and anti-inflammatory properties, data obtained from both in vitro and in vivo studies suggest that statins also have antiproliferative, antiangiogenic, and anti-metastatic properties. Statins may thus represent a novel therapeutic approach for cancer prevention and treatment.

Several important clinical questions remain to be addressed about the role of statins in cancer patients. First, the tumor types most susceptible to statin therapy have yet to be determined. Current pre clinical and clinical data indicate that statins might be potentially useful for the treatment of melanoma (37), leukemia, brain cancer (38,39), hepatocellular cancer (124) and squamous cell cancer of the head and neck (43,59). Second, it is not known which statins are most effective for cancer prevention and treatment. Although both hydrophilic (e.g., pravastatin and rusovastatin) and lipophilic (e.g., simvastatin and atorvastatin) statins demonstrate antitumor effects, hydrophilic statins are, in general, thought to be less effective intra cellular agents.

Nonetheless, hydrophilic statins, such as pravastatin, are associated with lower risks for colorectal (103) and hepatocellular cancer (41). Third, the optimal statin regimen has yet to be defined. Statins alone are not effective anticancer agents. However, when combined with other cytotoxic agents, preclinical and clinical data suggest that statins may enhance chemotherapeutic effects. Most clinical trials to date have favored continuous, low-dose statin regimens over brief, high-dose statin regimens. This has been largely a result of the fact that low-dose regimens are associated with a lower risk for statin side effects, including myopathy, rhabdomyolysis, and hepatotoxicity. Moreover, statin side effects are more likely to occur in cancer patients, because they often receive concomitant medications, such as erythromycin and cyclosporine, which are known to interfere with statin metabolism through the hepatic cytochrome P-450 system (125). In summary, statins may be beneficial for the prevention and treatment of cancer. Now is the time for appropriately designed clinical trials to underpin the promising data of pre-existing studies.

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XXIII - NONSTEROIDAL ANTIINFLAMMATORY DRUGS AS ANTICANCER AGENTS: MECHANISTIC, PHARMACOLOGIC, AND CLINICAL ISSUES A. Ferreira-Alemao, MD PhD UNIVERSIDAD COMPLUTENSE DE MADRID

Numerous experimental, epidemiologic, and clinical studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs), particularly the highly selective cyclooxygenase (COX)-2 inhibitors, have promise as anticancer agents. NSAIDs restore normal apoptosis in human adenomatous colorectal polyps and in various cancer cell lines that have lost adenomatous polyposis coli gene function. NSAIDs also inhibit angiogenesis in cell culture and rodent models of angiogenesis. Many epidemiologic studies have found that long-term use of NSAIDs is associated with a lower risk of colorectal cancer, adenomatous polyps, and, to some extent, other cancers.

Two NSAIDs, sulindac and celecoxib, have been found to inhibit the growth of adenomatous polyps and cause regression of existing polyps in randomized trials of patients with familial adenomatous polyposis (FAP).

However, unresolved questions about the safety, efficacy, optimal treatment regimen, and mechanism of action of NSAIDs currently limit their clinical application to the prevention of polyposis in FAP patients.

Moreover, the development of safe and effective drugs for chemoprevention is complicated by the potential of even rare, serious toxicity to offset the benefit of treatment, particularly when the drug is administered to healthy people who have low annual risk of developing the disease for which treatment is intended. This review considers generic approaches to improve the balance between benefits and risks associated with the use of NSAIDs in chemoprevention. We critically examine the published experimental, clinical, and epidemiologic literature on NSAIDs and cancer, especially that regarding colorectal cancer, and identify strategies to overcome the various logistic and scientific barriers that impede clinical trials of NSAIDs for cancer prevention. Finally, we suggest research opportunities that may help to accelerate the future clinical application of NSAIDs for cancer prevention or treatment.

Several recent reviews (1-3) have summarized the intriguing and accumulating evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) have promise as anticancer drugs. NSAIDs have been shown experimentally to stimulate apoptosis and to inhibit angiogenesis, two mechanisms that help to suppress malignant transformation and tumor growth. Numerous epidemiologic (nonrandomized) studies ( 4-47) have found that long-term users of aspirin or other NSAIDs have a lower risk of colorectal adenomatous polyps and colorectal cancer than nonusers, although one study has not ( 38-40). Randomized clinical trials have confirmed that two NSAIDs, the prodrug sulindac ( 41-43) and the selective cyclooxygenase (COX)-2 inhibitor celecoxib (44), effectively inhibit the growth of adenomatous polyps and cause regression of existing polyps in patients with the unusual hereditary condition familial adenomatous polyposis (FAP).

Despite these positive findings, the efficacy and safety of long-term NSAID prophylaxis against colorectal or other cancers remain unproven. While some experts have proposed that there is now sufficient evidence for persons at high risk of large bowel cancer to begin taking low-dose aspirin prophylactically ( 45), most have not. Health organizations and consensus groups have been appropriately cautious by withholding any recommendation regarding the use of NSAIDs for the prevention or treatment of cancer, except for the use of celecoxib or sulindac to suppress the growth of colorectal adenomatous polyps in patients with FAP ( 46).

Despite enthusiasm about the potential usefulness of NSAIDs, particularly the selective COX-2 inhibitors, as 162

RIBOGRAMA PROJECT anticancer agents, fundamental questions remain about their safety, efficacy, mechanisms of action, optimal treatment regimens, and contraindications for preventive therapy.

Because of the formidable challenges involved in developing safe and effective drugs for chemoprevention, discussed below, there is continuing need to improve cross-disciplinary communication in planning randomized clinical trials of NSAIDs for chemoprevention. Using the example of aspirin prophylaxis for the prevention of cardiovascular disease, we consider the delicate balance of risks and benefits that complicates primary prevention of cancer. The safety of treatment is of particular concern when large numbers of healthy people must be treated prophylactically for many years to prevent adverse events in a small percentage of those treated. We suggest generic strategies that can improve the benefitâ&#x20AC;&#x201C;risk balance. We critically review the published experimental, clinical, and epidemiologic evidence regarding NSAIDs and cancer, focusing particularly on colorectal cancer, and suggest research strategies and opportunities that may help to accelerate the future clinical application of NSAIDs for the prevention or treatment of cancer.

CHALLENGES OF CHEMOPREVENTION At least two constraints make it particularly difficult to develop safe and effective drugs to prevent cancer in average- or low-risk populations. The first is the very low margin for toxicity whenever prophylactic drugs are administered over a long period of time to healthy people who have comparatively low risk of the disease being prevented. Although colorectal cancer accounts for almost 12% of all newly diagnosed cancers in the United States (47), the probability that an individual at average risk will develop colorectal cancer in a given year is low. On average, the cumulative probability of developing colorectal cancer from birth to age 79 years is approximately 4% in men and 3% in women; this probability increases to 5.6% in both sexes over full-life expectancy (48). Thus, in the general population, more than 94% of the people treated prophylactically to prevent colorectal cancer will not benefit from prophylactic treatment unless the benefits extend to other health endpoints. However, the risk of colorectal cancer is considerably higher in certain genetically susceptible subgroups. For example, estimates of lifetime risk, based on studies of high-risk families, are approximately 17% for persons with two affected first-degree relatives, 70% for individuals with genetic mutations associated with hereditary nonpolyposis colon cancer (HNPCC), and more than 95% in persons with FAP ( 49). However, these high-risk groups contribute only a small fraction of all cases of colorectal cancer.

A second constraint to developing drugs to prevent cancer in average- or low-risk populations concerns the logistic difficulty of studying cancer endpoints, especially those associated with colorectal cancer, in largescale prevention trials. Because of the low annual risk of developing colorectal cancer in the general population, phase III cancer prevention trials must be much larger than either trials of cardiovascular disease in high-risk populations or therapeutic trials in cancer patients already diagnosed with disease to have adequate statistical power. More than 150 clinical trials have been conducted to assess the efficacy of aspirin as an antiplatelet agent in patients at high risk of cardiovascular disease. Most of these trials enrolled fewer than 3000 patients, and follow-up ranged from 6 months to 4 years (50,51). Trials of similar size and duration have sufficient statistical power to assess the prevention of recurrent colorectal adenomatous polyps in highrisk groups. In contrast, trials of colorectal cancer prevention would require up to 100 times more subjects with 10â&#x20AC;&#x201C;20 years of follow-up. Furthermore, trials of both adenomatous polyps and colorectal cancer prevention are logistically more difficult than prevention trials of other cancer sites because they require that study subjects undergo special examinations (i.e., sigmoidoscopy or colonoscopy) before and after the intervention.

Consequently, clinical decisions about the efficacy of chemopreventive drugs will need to be based on a smaller number of phase III trials that measure surrogate endpoints in high-risk populations. Decisions about the treatment regimen to be tested in these trials must be based largely on preclinical and epidemiologic evidence, on phase I trials involving 20â&#x20AC;&#x201C;80 participants, and on phase II studies of up to several hundred subjects.

163

RIBOGRAMA PROJECT EXAMPLE OF ASPIRIN IN THE PREVENTION OF CARDIOVASCULAR EVENTS Randomized trials of aspirin in the prevention of cardiovascular disease illustrate that the safety of prophylactic treatment is influenced critically by the background risk of the population being treated ( 50,52,53).

Approximately three additional adverse events are prevented for every 10% increase in annual background risk. The largest potential benefit, occurs during the month immediately following an acute MI, wherein 1 month of aspirin treatment prevents approximately 37 vascular events (MI, stroke, or death from all causes) per 1000 persons treated ( 50,52). The cardiovascular benefit of aspirin prophylaxis is smallest in low-risk populations, such as healthy male doctors, in which only 0.09 events are prevented per 1000 patient-months of treatment (56).

Cardiovascular benefit of aspirin in relation to background cardiovascular risk. The x-axis indicates the percentage of placebo-treated subjects experiencing an adverse vascular event each year. The y-axis indicates the number of adverse cardiovascular events (myocardial infarction [MI], stroke, or death from all causes) prevented per 1000 patient-months of treatment with aspirin at less than or equal to 500 mg daily (50,54-56).Each point (and 95% confidence interval [CI]) represents the combined data from all published randomized trials of aspirin prophylaxis at less than or equal to 500 mg daily in the specified population. By comparison, the mean frequency of serious bleeding complications in aspirin trials at this dose is 0.07 per 1000 patient-months of treatment (95% CI = 0.04 to 0.09), with a maximum frequency of 0.65. TIA = transient ischemic attack.

The principal adverse effect of aspirin at doses of less than or equal to 500 mg daily is a small increase in the risk of serious bleeding complications. The mean frequency of hemorrhagic complications, per 1000 patientmonths of treatment, is 0.07 (95% CI = 0.04 to 0.09). The maximum frequencies are 0.64 and 0.65 in patients undergoing valve surgery and in those with acute MI, respectively. Within each population, the risk of serious bleeding varies with age and comorbidity ( 57). Serious bleeding complications are sufficiently rare that the risk is offset by potential cardiovascular benefits among patients whose background cardiovascular risk exceeds 3% annually. The risk–benefit balance is equivocal among intermediate-risk patients (i.e., those with a 1%–3% annual incidence of adverse cardiovascular events) and unfavorable among low-risk populations (i.e., those with an annual incidence of <1%). Clinicians should be aware that the calibration of risks and benefits associated with aspirin prophylaxis remains imprecise for any particular patient profile and that prudent treatment decisions require some margin for uncertainty. While aspirin prophylaxis has become standard therapy for patients with diverse high-risk cardiovascular conditions, it has not yet been proven to provide a net clinical benefit in intermediate- or average-risk settings (52).

GENERAL MECHANISM OF ACTION OF NSAIDS The mechanism of action that defines NSAIDs as a class is their ability to inhibit the COX activity of the enzyme prostaglandin G/H-synthase and thereby block the biosynthesis of prostaglandins ( 58). NSAIDs prevent the formation of prostaglandin H2, the first committed step in the metabolism of arachidonic acid into a complex cascade of signaling lipids, such as prostaglandin D2, prostaglandin E2, prostaglandin F2α, prostaglandin I2, and thromboxane, the principal prostanoid metabolite in platelets. Therapeutic concentrations of NSAIDs (usually in the low micromolar range) are not known to influence other pathways of arachidonic acid metabolism except indirectly by increasing the intracellular concentration of free arachidonic acid, which potentially causes shunting of arachidonic acid through other metabolic pathways.

The major metabolites of arachidonic acid produced by the cyclooxygenase (COX) and lipoxygenase (LO) pathways are indicated. Examples of tissues in which individual prostanoids exert prominent effects are indicated in parentheses. PGD2 = prostaglandin D2; PGE2 = prostaglandin E2; PGF2α = prostaglandin F2α; 164

RIBOGRAMA PROJECT TXA2 = thromboxane A2; PGI2 = prostaglandin I2; HPETE = hydroxyperoxyeicosatetraenoic acid; HETE = hydroxyeicosatetraenoic acid.

Two distinct isoforms of prostaglandin G/H-synthase, designated COX-1 and COX-2, have been recognized since 1991 (59,60). COX-1 is expressed constitutively in many tissues, and it plays a central role in platelet aggregation and gastric cytoprotection (61,62). Although COX-2 is expressed constitutively in the human kidney and brain, its expression is induced in many tissues during inflammation, wound healing, and neoplasia. COX-1 and COX-2 initiate the formation of biologically important prostanoids that coordinate signaling between the cell of origin (autocrine) and neighboring cells (paracrine) by binding to transmembrane G-protein-coupled receptors (61).

NSAIDs vary in their abilities to inhibit COX-1 or COX-2 at different concentrations and in different tissues. For example, aspirin is a relatively selective inhibitor of COX-1 in platelets when given at doses of 50â&#x20AC;&#x201C;100 mg daily (64,65) but inhibits COX-2 only at plasma concentrations higher than 0.5 mM. Most other conventional NSAIDs, such as ibuprofen, sulindac, and indomethacin, inhibit COX-1 and COX-2 to the same extent, whereas a new class of NSAIDs, designated coxibs by the World Health Organization, selectively inhibits COX-2 (66,67). Coxibs were developed to suppress prostanoid formation by COX-2 in inflammation while sparing the protective effects of COX-1 and its products prostaglandin I2 and prostaglandin E2 on gastric epithelium. Nevertheless, the availability of coxibs has stimulated research on the role of COX-2 in neoplasia and the potential efficacy and safety of selective COX-2 inhibition against cancer.

The pharmacologic effects of NSAIDs are further complicated by the diverse functions of prostanoids in different tissues and by the variable effects of COX inhibition, depending on drug, dose, and clinical context.

The formation of specific prostanoids varies across different tissues because of differences in the concentration of tissue-specific isomerases that catalyze their production from prostaglandin H2. Furthermore, more than one G-coupled protein receptor may transduce different effects from the same prostanoid ( 63). The diverse effects of COX inhibition can also be either therapeutic or deleterious, depending on the clinical characteristics of the patient. For example, low-dose aspirin (30â&#x20AC;&#x201C;150 mg daily) selectively inhibits the production of thromboxane A2 in platelets by COX-1, thereby suppressing platelet aggregation, vasoconstriction, and hemostasis ( 64,65). This antithrombotic effect may be beneficial for the majority of patients at high risk of occlusive vascular disease but deleterious for those who develop bleeding complications. Anti-inflammatory doses of NSAIDs (e.g., ibuprofen at a dose of 800 mg every 8 hours or naproxen at a dose of 500 mg twice daily) are therapeutic for patients with osteoarthritis or rheumatoid arthritis but can cause gastrointestinal ulceration, bleeding, or disruption of renal hemodynamics in susceptible patients.

A current controversy concerns whether long-term use of coxibs, which minimize serious gastrointestinal toxicity, may promote thrombosis or offset the cardiovascular benefits of low doses of aspirin by suppressing prostacyclin in vascular endothelial cells (63). Although coxibs do decrease urinary excretion of prostacyclin metabolites in normal subjects ( 69-71), currently available data do not resolve whether prolonged suppression of COX-2 may adversely affect thrombosis. Given the biologic complexity of prostanoid metabolism, however, it is not surprising that drugs that inhibit the activity of COX isoenzymes can have untoward as well as desirable effects on human health.

STRATEGIES TO IMPROVE THE SELECTIVITY OF NSAIDS Several generic strategies have been developed to improve the selectivity and reduce the toxicity of NSAIDs. One strategy is to identify high-risk populations in which the benefits of treatment would outweigh any attendant toxicity. A second strategy, which is taken by pharmaceutical companies, is to develop novel coxibs that inhibit COX-2 and suppress inflammation in chronic arthritis patients while sparing COX-1, thus avoiding the most serious gastrointestinal toxic effects ( 67,72). Currently available drugs with these properties are 165

RIBOGRAMA PROJECT celecoxib and rofecoxib. Other highly selective COX-2 inhibitors, such as valdecoxib, etoricoxib, and COX189, are now completing phase III trials of efficacy and safety in patients with osteoarthritis and rheumatoid arthritis.

A third approach to improve the selectivity and reduce the toxicity of NSAIDs is to determine the lowest effective drug dose and the most critical period for administration to achieve a specific pharmacologic effect.

For example, low-dose aspirin (50â&#x20AC;&#x201C;100 mg daily) is as effective as aspirin at higher doses in inhibiting COX-1 activity in platelets (64,65) and preventing MI and thrombotic stroke ( 52) while minimizing gastrointestinal toxicity. Decisions about the optimal treatment regimen and lowest effective dose are more easily made when the mechanism of action is known. For example, aspirin is the only NSAID that covalently and irreversibly inactivates COX-1, and it does this at concentrations that reach platelets in the enterohepatic circulation but are largely metabolized by the liver before entering the systemic circulation ( 53). Platelets lack a nucleus and, therefore, cannot synthesize new COX-1 during their 7- to 10-day life span. This combination of factors allows once-daily use of low-dose aspirin to provide full protection against platelet aggregation, despite its half-life of approximately 20 minutes in the systemic circulation (53).

A fourth strategy to improve the balance of benefits and risks associated with NSAID use is to identify combinations of drugs that are effective at very low doses ( 73,74). For example, much lower doses of sulindac and the cholesterol-lowering drug lovastatin are required to suppress chemically induced cancer in rodents and to stimulate apoptosis in human tumor cells when the drugs are given simultaneously than when either drug is given alone (75). Besides lovastatin, a 3-hydroxy-3-methylglutaryl coenzymeA reductase inhibitor, other drugs that have also been used in combination with either sulindac ( 73), aspirin (76), or piroxicam (77,78) include ornithine decarboxylase inhibitors (76-78), the spice curcumin (79), and EKI785, an irreversible inhibitor of the epidermal growth factor receptor kinase ( 73).

EVIDENCE FOR CANCER PREVENTION PROPERTIES OF NSAIDS CHEMICALLY INDUCED INTESTINAL CANCER IN RODENTS The hypothesis that NSAIDs might inhibit the occurrence or growth of colorectal cancer arose in the mid1970s, when Bennett and Del Tacca (80) and Jaffe (81) reported that the concentration of prostaglandin E2 was higher in human colorectal tumor tissue than in the surrounding normal mucosa. These relatively crude measurements of prostaglandin concentrations in human tumors stimulated more than 40 experiments in which numerous NSAIDs were shown to inhibit chemically induced colorectal cancer or aberrant crypt formation in rats or mice (76-79,82-116). In these animal models, weanling rats or mice were given a subcutaneous injection of azoxymethane or other carcinogens known to induce intestinal cancer and were subsequently given known concentrations of NSAIDs in their food or water. Those experiments varied the timing of the NSAID treatment in relation to the carcinogen exposure by initiating treatment before exposure to the carcinogen (initiation phase), during the promotion-progression phase, or both. Colorectal tumors produced in the rat model share many characteristics with human colorectal cancer, except the former have a lower tendency to metastasize (117).

The studies in rodents proved conclusively that aspirin, other conventional NSAIDs (such as piroxicam, indomethecin, sulindac, ibuprofen, and ketoprofen), and selective COX-2 inhibitors [e.g., celecoxib (107)] inhibit chemically induced carcinogenesis in rats and mice ( 46). The highest tolerated dose of nonselective NSAIDs typically reduced the number and size of tumors by 40%â&#x20AC;&#x201C;60%. Nonselective NSAIDs suppressed but did not completely eliminate the growth of chemically induced adenomatous polyps and cancers. Two studies of coxibs in this model (107,114) have indicated that high doses of celecoxib (1500 ppm in food) inhibit 90% of tumors in rats and are better tolerated than comparable doses of nonselective NSAIDs. At least three important insights can be derived from the rodent experiments. First, nonselective NSAIDs suppress tumor growth to a greater extent and at lower doses when treatment is begun before or coincident 166

RIBOGRAMA PROJECT with exposure to the carcinogen than when it is delayed until the tumor promotion/progression phase. For example, low-dose piroxicam (25 ppm in food) caused a 30% reduction in tumors when treatment was begun soon after exposure to the carcinogen but only a 12% reduction when treatment was begun 23 weeks after exposure (98). Early initiation of treatment also improves tumor suppression by sulindac sulfone (110) and celecoxib (118). Second, both nonselective and selective NSAIDs effectively inhibit the early stages of tumor development, whereas only selective COX-2 inhibitors are effective when treatment is delayed. For example, celecoxib (1500 ppm in food) reduced tumor incidence and multiplicity by approximately half, even when treatment was delayed until the tumor promotion/progression stage ( 118). Third, NSAID treatment must be continued without interruption to prevent resumption of tumor growth (91,92).

Experimental studies in rodents have several advantages not offered by other types of studies. First, the ability to administer measured concentrations of single NSAIDs to intact animals provides a greater opportunity to examine complex interactions within and across cells than exists in studies of isolated cell cultures and other in vitro models. Second, the rodent experiments are not subject to confounding by lifestyle factors, which may afflict epidemiologic studies. Limitations of the rodent experiments, other than potential interspecies differences, include a lack of uniformity in experimental design that limits comparisons across studies and the lack of measurements of NSAID concentrations in blood, COX-1 activity in platelets, or COX-2 activity in activated monocytes. Measurements of COX-2 activity in activated monocytes could help to identify the enzymatic target involved in tumor inhibition.

Other experimental studies indicate that NSAIDs inhibit many induced and transplanted cancers in various animal models, although the evidence for this is more limited than that for colorectal cancer. Other cancers potentially affected by NSAIDs include tumors of the esophagus(119-121), stomach (122,123), skin (124), breast (125-128), lung (129-132), prostate (133,134), and urinary bladder (135-137). A comprehensive review of these studies is beyond the scope of this review.

CLINICAL STUDIES AND RANDOMIZED TRIALS OF NSAIDS IN FAP Randomized clinical trials have established that two NSAIDs, sulindac (41,42) and celecoxib (44), suppress adenomatous polyps and cause regression of existing polyps in patients with FAP. FAP is a rare hereditary condition resulting from germline inactivation of one allele of the adenomatous polyposis coli (APC) gene.

Affected individuals develop tens to thousands of adenomatous polyps. If these individuals do not undergo surgical resection of the colon, virtually all develop colorectal cancer by the third or fourth decade of life ( 138).

FAP accounts for only 1% of human colorectal cancers, yet it provides a model of APC inactivation as an early genetic event for the approximately 85% of cancers that develop from sporadic adenomatous polyps.

Treatment with both sulindac and celecoxib is used to supplement surgery in FAP patients (139) However, it should be noted that some FAP patients have developed rectal carcinoma, despite ongoing therapy with sulindac (140,141) and that adenomatous polyps resume growth in FAP patients if NSAID prophylaxis is stopped. The one published study that evaluated NSAID prophylaxis in relation to the regression of small (<1 cm) sporadic adenomatous polyps ( 14,21,42) found no statistically significant difference in polyp size among the 18 patients treated with sulindac (300 mg) for 4 months.

MOUSE MODELS OF FAP Several murine models that resemble human FAP have been developed and used to determine whether various NSAIDs and coxibs suppress the development of spontaneous intestinal adenomas (143).

Nonselective NSAIDs, such as piroxicam ( 144), sulindac (145-147), and aspirin (148), and selective COX2 inhibitors, such as celecoxib (144) and rofecoxib (149), inhibit tumor development in ApcMin mice and other murine models of FAP. These models mimic the rapid development of adenomatous polyps that affects 167

RIBOGRAMA PROJECT humans with germline inactivation of one APC gene but differ from FAP in that the mouse tumors occur predominantly in the small intestine.

OBSERVATIONAL EPIDEMIOLOGIC STUDIES IN THE GENERAL POPULATION Numerous (nonrandomized) epidemiologic studies have reported that people who regularly use aspirin and other NSAIDs have a lower incidence of adenomatous polyps and lower incidences of or deaths from colorectal cancer compared with nonusers (4-10). The consistency of these findings is striking, despite different researchers using various study designs in diverse patient populations. Sustained NSAID use is associated with a 30%â&#x20AC;&#x201C;50% reduction in adenomatous polyps, incident disease, and death from colorectal cancer in all but one of these epidemiologic studies ( ). Epidemiologic studies of the association between nonsteroidal anti-inflammatory drug (NSAID) use and colorectal cancer or adenomatous polyps. The relative risk estimates (circles) and 95% confidence intervals (lines) refer to the incidence or death rate among regular NSAID users compared with that among nonusers in A) cohort studies, B) caseâ&#x20AC;&#x201C;control studies of NSAIDs and colorectal cancer, and C) studies of NSAIDs and adenomatous polyps. Results from these studies strongly support the hypothesis that NSAIDs inhibit the occurrence and/or progression of colorectal cancer in the general population, not just in FAP patients ( 150,151). The aggregate findings cannot be attributed to earlier detection because of aspirin-induced bleeding or measured potential confounders. Besides attracting substantial research interest and funding to the NSAID hypothesis, the results of these epidemiologic studies suggest that the duration and continuity of NSAID use may be more critical than the daily dose ( 12,15). Like the rodent experiments and clinical studies of FAP, the epidemiologic studies also suggest that tumors resume growth after termination of NSAID treatment ( 19,30).

Epidemiologic studies cannot, however, provide randomized evidence that NSAIDs prevent the development of adenomatous polyps or cancer nor have past analyses fully defined the optimal drug, dose, treatment regimen, age to begin prophylactic therapy, or the balance of risks and benefits in different patient populations.

Nevertheless, these studies raise an intriguing mechanistic question in that the dose and dosing frequency of aspirin associated with lower risk of colorectal cancer are often insufficient to sustain COX-2 inhibition in nucleated cells (6,10). The possibility that activated platelets may contribute to the induction of COX-2 is discussed below. Other epidemiologic studies have found that prolonged use of NSAIDs is associated with lower incidence of or deaths from cancers at several other sites. The literature regarding these other cancers includes studies of tumors of the esophagus ( 7), stomach (7), breast (5,7,9,16,38,153-157), lung (7), prostate (158), urinary bladder (7), and ovary (7,159-161). However, there are fewer studies of these other cancers than of colorectal cancer and the results are less consistent.

COMPLETED RANDOMIZED TRIALS IN THE GENERAL POPULATION The Physicians' Health Study is the only randomized clinical trial of aspirin in the primary prevention of cardiovascular endpoints of sufficient size to measure incidence or death rates from colorectal cancer, even though the aspirin arm of this trial was terminated after 5 years ( 56). This study showed no reduction in either invasive or in situ colorectal cancer incidence nor a reduction in colorectal cancer mortality among 22 071 male physicians who were randomly assigned to receive either 325 mg of aspirin or placebo every other day for 5 years, with a 12-year follow-up (56). However, interpretation of these results is limited because of the short duration of randomized treatment, the lack of systematic screening for adenomatous polyps or cancer at the beginning and end of the trial, and the relatively low dose of aspirin tested.

MECHANISTIC STUDIES OF NSAIDS AND APOPTOSIS.

168

RIBOGRAMA PROJECT Despite continuing uncertainty about the molecular pathways by which NSAIDs may inhibit colorectal neoplasia, there is mounting evidence that tumor inhibition may be mediated by at least two distinct cellular processes. These involve the ability of NSAIDs to restore apoptosis in APC-deficient cells (162,163) and their capacity, particularly in the case of coxibs, to inhibit angiogenesis.

Apoptosis, or programmed cell death, is needed to maintain homeostasis in continuously replicating tissues such as the intestine (164). Partial suppression of apoptosis occurs early in tumorigenesis in approximately 85% of human colorectal cancers due to the inactivation of both alleles of the APC gene ( 165,166). The suppression of apoptosis allows APC-deficient cells to accumulate in adenomatous polyps. Further suppression of apoptosis occurs as these cells develop additional genetic mutations and phenotypic changes (167).

In vitro, both nonselective NSAIDs and selective COX-2 inhibitors stimulate apoptosis in APC-deficient cells that have not yet undergone malignant transformation. This is also seen clinically in FAP patients treated with sulindac (168) and in experimental studies of ApcMin mice ( 145,147,148,169) and rats exposed to chemical carcinogens (170). Nonselective NSAIDs lose their ability to inhibit chemically induced tumors when polyps undergo malignant transformation. In contrast, selective COX-2 inhibitors stimulate apoptosis and suppress growth in many carcinomas, including cultured human cancers of the stomach ( 171), esophagus (121,172), tongue (173), brain (174), lung (130), and pancreas (175).

The precise mechanism by which NSAIDs restore apoptosis remains controversial ( 179), although it clearly affects factors related to APC deficiency or the induction of COX-2 or both. Apoptosis can be suppressed in normal human or rodent intestinal epithelial cells by manipulating these cells to overexpress COX-2 (162,177). In human HT-29 colon cancer cells, apoptosis can be restored by treatment with selective ( 178) or nonselective (179,180) COX inhibitors or by restoring APC gene function ( 165). Apoptosis becomes progressively more inhibited during the development of colorectal cancer (167), coincident with the increasing expression of COX-2. For example, COX-2 is undetectable in normal epithelium but is detectable in 40% of adenomatous polyps (181) and in more than 80% of colorectal cancers. COX-2 expression in human colorectal carcinomas is associated with larger tumor size and deeper invasion, although not with metastases (182).

Other studies (183,184) suggest that COX-1 activity, perhaps through the induction of COX-2, may also be essential for the development of colorectal neoplasia. In mouse knockout studies ( 183,184), deletion of either the COX-1 or COX-2 genes in Apc-deficient mice caused a 70%â&#x20AC;&#x201C;80% reduction in intestinal polyposis.

We have hypothesized that COX-1 activity in activated platelets may signal the increased expression of COX2 in other cells through the release of lipid or protein paracrine mediators ( 185). A role for COX-1 in the induction of COX-2 might explain why, in epidemiologic studies, aspirin use is associated with reduced risk of colorectal cancer even at doses and dosing intervals that could not sustain COX-2 inhibition in nucleated cells (6,12).

Despite these observations, results from other studies challenge the conventional wisdom that COX inhibition is the only shared function of NSAIDs (186) or that the products rather than the substrate of COX activity mediate its biologic effects. For example, in some experimental models, the concentration of free arachidonic acid itself regulates apoptosis in colorectal epithelial cells (187,188). Other experimental models suggest that NSAIDs may affect apoptosis through a mixture of prostaglandin-dependent and prostaglandinindependent pathways (176). The selective COX-2 inhibitor NS-398 stimulates apoptosis in human S/KS colon carcinoma cells, which do not express COX-2 enzyme, as well as in HT-29 cells, which do (178).

Although sulindac sulfone is believed not to inhibit COX activity, it nonetheless stimulates apoptosis in rats exposed to chemical carcinogens ( 110) and in human HT-29 colon carcinoma cells (189). High concentrations of sulindac sulfone and sodium salicylate reportedly modify signal transduction through either 169

RIBOGRAMA PROJECT the c-MYC oncogene (190), nuclear factor-κB (191,192), or p38, a mitogen-activated protein kinase (193,194). Very high concentrations of sulindac sulfide inhibit transcriptional activation by the nuclear peroxisome proliferator-activated receptor-δ (163), a nuclear hormone receptor regulated partly by APC gene function (163,195). NSAIDs have also been reported to induce apoptosis through 15-lipoxygenase-1, independent of COX-2 (196). However, many of these effects have been demonstrated only with high concentrations of NSAIDs in vitro and are of uncertain clinical relevance.

MECHANISTIC STUDIES OF ANGIOGENESIS. A second cellular process by which COX-2 inhibitors may inhibit tumor growth is through inhibition of angiogenesis and neovascularization (197,198). Solid tumors must stimulate the formation of new capillary blood vessels to grow larger than approximately 2 mm in diameter ( 198-200). COX-2 expression is widely induced in the angiogenic vasculature of colorectal adenomatous polyps and in carcinomas of the colon, lung, breast, esophagus, and prostate ( 200,201). Selective COX-2 inhibitors suppress the growth of corneal capillary blood vessels in rats exposed to basic fibroblast growth factor ( 201) and inhibit the growth of several human tumors transplanted into mice (201,201). Therapeutic (low micromolar) concentrations of coxibs also suppress the release of angiogenic growth factors by human or rodent colorectal cancer cells that are cocultured with vascular endothelial cells (197,198) and block migration and tube formation by the endothelial cells. In contrast, toxic concentrations of aspirin ( 197) or indomethacin (198) are required to block vascular endothelial tube formation. These experiments suggest that COX-2 may be essential for tumor vascularization and growth. However, the relevance of the experimental models to human colorectal cancer remains uncertain.

IMPLICATIONS FOR ONGOING AND FUTURE SCIENTIFIC RESEARCH RANDOMIZED TRIALS OF NSAIDS AND PREVENTION OF COLORECTAL CANCER Approximately 20 randomized clinical trials designed to test whether nonselective NSAIDs or coxibs inhibit the early development of colorectal cancer either have been completed or are ongoing. Most are phase I and II studies to determine the bioactivity of various NSAID treatments in rectal epithelium and/or their gastric toxicity (203-206). We believe that the ongoing phase I studies could be strengthened by testing validated biomarkers of apoptosis and angiogenesis in adenomatous polyps and early-stage carcinomas. For example, quantitative measures of apoptosis in adenomatous polyps may be a more sensitive index of the biologic effect of NSAIDs than are measures of cell proliferation, aberrant crypt formation, or prostaglandin concentrations in normal rectal mucosa. Improved biomarkers are essential for phase I/II studies to identify the least toxic treatments and drug combinations for further testing.

Seven phase III trials are currently testing whether aspirin, other nonselective NSAIDs, or selective COX-2 inhibitors suppress the development of adenomatous polyps or cancer in high-risk patients. These prevention trials will provide the first randomized evidence of the effectiveness of aspirin at doses of 80–300 mg daily or celecoxib at doses of 200–400 mg daily in inhibiting the development of sporadic adenomatous polyps in patients without FAP. Two of the studies are also testing whether aspirin and celecoxib inhibit colorectal cancer among patients with HNPCC, a condition that accounts for approximately 15% of colorectal cancers.

Measuring surrogate endpoints in high-risk populations improves the feasibility of cancer prevention studies, but it also introduces new sources of uncertainty. The prevention of adenomatous polyps is not synonymous with the prevention of colorectal cancer, nor are findings in patients with FAP or HNPCC necessarily generalizable to patients with most colorectal cancers. Furthermore, even if NSAIDs do reduce the risk of colorectal cancer, none of these trials will address the safety of administering prolonged treatment 170

RIBOGRAMA PROJECT prophylactically to healthy people. However, the evaluation of appropriately validated biomarkers ( 164) could strengthen the ability of phase III trials to test focused mechanistic hypotheses by verifying that the intervention being tested achieves the desired pharmacologic effect under field conditions.

TRIALS OF NSAIDS AS ADJUVANT THERAPY New trials are testing the efficacy of coxibs to treat precancerous lesions of the mouth, esophagus, and skin and as adjuvant therapy for solid tumors that express COX-2 (172,175,207-211). Therapeutic trials are easier to conduct and are more cost-effective than prevention trials because of their smaller size and shorter follow-up (201). Therapeutic trials can directly measure clinical endpoints, such as tumor recurrence and survival, as well as potential surrogate measures of disease. Furthermore, the high risk of cancer recurrence or progression in therapeutic settings offsets some of the constraints on toxicity that limit the prevention trials.

A strong scientific rationale supports therapeutic trials of coxibs for the treatment of cancers that express COX2 in vascular endothelium (172,175,207-211). First, celecoxib suppresses the growth of human colon and lung tumors xenografted into rodents (212). Second, adjuvant treatment with coxibs enhances the response of human HT-29 colon cancer cells and mouse sarcoma transplants to standard chemotherapy or radiation therapy (213,214). Third, a cross-sectional clinical study has shown that COX-2 expression in human colorectal tumors is directly associated with tumor stage and size at diagnosis and is inversely associated with patient survival (215). Evidence from randomized trials suggesting that coxibs inhibit recurrence or prolong survival in early-stage colorectal cancer would stimulate further trials of adjuvant treatment of other COX-2expressing solid tumors.

FUTURE ROLES FOR EPIDEMIOLOGIC STUDIES An important continuing role for epidemiologic studies is to quantify the benefits and risks of NSAID treatment across a broader range of treatment regimens, outcomes, and patient populations than is feasible in randomized clinical trials. Whereas epidemiologic studies can assess multiple endpoints and prolonged exposures, randomized studies must generally be stopped when benefit or toxicity is demonstrated conclusively. Epidemiologic studies can assess how cofactors, such as age and comorbidity, affect the likelihood of benefits and risks in different patient populations. Ultimately, clinical guidelines should enable a practicing physician to consider how NSAIDs affect multiple health endpoints (cancer, cardiovascular, and other) in specific patient populations.

BASIC RESEARCH Studies of chemically induced colorectal cancer in rats have established that numerous NSAIDs inhibit colorectal neoplasia in this model. Studies of whole animals will continue to be important for identifying the cellular targets and molecular pathways by which this inhibition occurs. Epithelial cells do not exist in a vacuum. Research on whole animals reveals the complex interactions that occur between adjoining tissues or between epithelial cells and the underlying stroma ( 216). The development of drugs that are more selective and effective than those currently available will require a better understanding of the complex intracellular signaling that occurs in intact animals but may not be replicated by cell culture or other laboratory models (217,218).

Studies using transgenic animals can also identify the genetic and epigenetic effects of NSAIDs. Deletion of the genes that code for enzymes (e.g., inducible prostaglandin E2 synthase) and receptors (e.g., the thromboxane receptor) downstream of prostaglandin G/H synthase may help to characterize the lipid mediator(s) involved in the modulation of apoptosis and angiogenesis. Additional mechanistic insights can be 171

RIBOGRAMA PROJECT obtained by measuring NSAID concentrations in plasma, COX-1 activity in circulating platelets, and COX-2 activity in activated monocytes.

We hope that the issues raised by this review will help to sustain progress in this exciting area.

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XXIV - EXTRACOLONIC TUMOURS IN PATIENTS WITH LYNCH II SYNDROME A. Ferreira-Alemao, MD PhD UNIVERSIDAD COMPLUTENSE DE MADRID

Introduction Hereditary nonpolyposis colorectal cancer, or Lynch syndrome, is responsible for 2â&#x20AC;&#x201C;3% of all colorectal cancers. Lynch syndrome is also associated with a high risk of extracolonic cancers, including endometrial, stomach, small bowel, pancreas, biliary tract, ovary, urinary tract, brain, and skin cancer. In this review, we discuss the risks, surveillance tests, and guidelines for the management of extracolonic tumours associated with Lynch syndrome.1 For all types of extracolonic cancer, evidence supporting surveillance is scarce. A benefit of surveillance is evident only for endometrial cancer, where transvaginal ultrasound and endometrial sampling detect tumours in early stages. Surveillance is generally recommended for urinary tract and gastric cancer, especially in families with more than one member with these types of cancer. For the other types of cancer, surveillance is typically not recommended. Prophylactic hysterectomy and bilateral salpingooophorectomy should be considered for women with Lynch syndrome who are past childbearing age, especially during surgery for colorectal cancer. No data show efficacy of chemopreventive drugs in reducing the risk of extracolonic cancers for patients with Lynch syndrome.

Lynch syndrome, or hereditary non-polyposis colorectal cancer, is the most common hereditary colon cancer and is characterized by a predisposition to early onset colorectal cancer and several extracolonic malignancies. In the past, diagnosis was based on family history and clinical criteria, such as the Amsterdam criteria. Identification of susceptibility genes for Lynch syndrome narrowed the diagnosis to families with pathogenic germline mutations in one allele of the mismatch repair genes MLH1, MSH2, MSH6, or PMS2.

Mutations in mismatch repair genes on both alleles cause a rare form of childhood onset cancer that we do not discuss. Disrupted mismatch repair function leads to replication errors in repetitive DNA segments, known as microsatellites. Microsatellite instability also occurs in some sporadic cancers.

The most common malignancy in individuals with Lynch syndrome is colorectal cancer, with a cumulative lifetime risk of up to 70% at age 70 years. People who are carriers of mutations in mismatch repair genes also have a risk of extracolonic cancers; these include cancer of the endometrium, ovary, stomach, urinary tract, biliary tract, pancreas, small bowel, brain, and skin. Carcinogenesis and biological behaviour of colorectal tumours differ in individuals with and without Lynch syndrome. Whether the same is true for other cancers associated with Lynch syndrome is unknown.

Surveillance is done in asymptomatic individuals to diagnose malignant or premalignant lesions at an early stage to improve survival. For people with Lynch syndrome, colorectal surveillance via colonoscopy is recommended every 1â&#x20AC;&#x201C;2 years beginning at age 20â&#x20AC;&#x201C;25 years. This strategy has reduced the incidence of and mortality from colorectal cancer.2,3 Surveillance of all other organs in which tumours might develop is not realistic with current methods. It is unclear for which extracolonic cancer types surveillance is benefi cial, at what age to start, and how often. In this systematic review, we outline the risks, surveillance tests, and guidelines for the management of extracolonic malignancies in Lynch syndrome.

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Risk of extracolonic malignancies Finding the best surveillance strategy for a tumour type first requires knowledge of the lifetime risk and age distribution at the time of diagnosis. Cancer risk is affected by external factors, such as diet and lifestyle, and by internal factors, such as genetic characteristics. The risk of extracolonic tumours for people with Lynch syndrome depends on which mismatch-repair gene is mutated. Carriers of mutations in MLH1, MSH2, MSH6, and PMS2 have different cancer risks. However, most individuals with Lynch syndrome have mutations in either MLH1 or MSH2, and data on the risks associated with the other mismatch repair genes are limited.

Some studies report risks associated with particular genes and others present collective risks. Estimations of cancer risk in patients with Lynch syndrome are from both proven and assumed carriers of mutations. Men have a higher risk of (non-gynaecological) malignant diseases than do women.24–28 Mutations in genes not involved in mismatch repair can affect cancer risk in families with Lynch syndrome; 1 however, testing for these variants is not part of the clinical risk assessment for Lynch syndrome. Risks of malignancies in individuals with Lynch syndrome are often expressed as standardized-incidence ratios: observed incidence in people with Lynch syndrome relative to that in the general population.4–28 The epidemiology and surveillance strategy for each type of cancer are discussed in the following sections. Treatments for each tumour type do not differ from treatment of the sporadic forms and are not addressed.

Gynaecological tumours Epidemiology For women with a genetic predisposition for Lynch syndrome, lifetime risk of endometrial cancer is higher than that of colorectal cancer. Women with Lynch syndrome have a 27–71% cumulative lifetime risk of endometrial cancer compared with 3% in the general population. Risk is between 27% and 60% for women with mutations in MLH1 and MSH2 and 60–71% for those with mutations in MSH6. Annual incidence of endometrial cancer in women with Lynch syndrome who are older than 40 years is 2.5%.29 Mean age at diagnosis for carriers of mutations in MLH1 and MSH2 is 59 years and 54 years for MSH6,8,17–19 although Vasen and colleagues 30 reported a younger age at onset. They studied 125 patients with endometrial cancer from families with Lynch syndrome and found a mean age at diagnosis of 48 years (range 27–72 years).30 Of all endometrial cancers, 57% were diagnosed before age 55 years and 98% before the age of 60 years.30 The lifetime risk of ovarian cancer in women with Lynch syndrome is around 7% (3–14%), compared with 1.4% in the general population.

In 72 women with Lynch syndrome, twice as many with mutations in MSH2 as with mutations in MLH1 had ovarian cancer, and the highest risk was in those aged 40–55 years.16 Endometrial carcinomas have the same histopathology in patients with and without Lynch syndrome.31,32 Cancers in the lower uterine segment seem to be associated with Lynch syndrome. In 35 of 1009 patients with endometrial cancer, the tumour was in the lower uterine segment.33 In unselected series of endometrial cancers, around 1.8% of cases are associated with Lynch syndrome. Further studies are needed to confirm these data. Most endometrial cancers associated with Lynch syndrome are detected at an early stage and have a good prognosis.

Ovarian tumours in women with Lynch syndrome are often non-serous.34 A recent meta-analysis found that ovarian tumours with mismatch-repair deficiency had earlier stage disease at presentation than did tumours without mismatch-repair deficiency.34 Only one study systematically investigated ovarian cancer survival in patients with Lynch syndrome compared with a registry-based control group; survival rates did not substantially differ.35 Strategies to lower the risks of endometrial and ovarian cancer in women with Lynch syndrome include surveillance, chemoprevention, and prophylactic surgery.

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RIBOGRAMA PROJECT Surveillance A high risk of endometrial cancer in women with Lynch syndrome and an easily-detectable premalignant stage are factors that support surveillance for endometrial cancer. These women are less aware of their risk for endometrial versus colorectal cancer and undergo endometrial cancer surveillance less often than colonoscopy.36 Generally, endometrial cancer diagnosed with transvaginal ultrasound in asymptomatic patients is associated with a better prognosis than when it is diagnosed at a symptomatic stage.37 A few studies have investigated the efficacy of gynaecological surveillance in women with suspected Lynch syndrome.38â&#x20AC;&#x201C;40 Dove-Edwin and co-workers38 retrospectively assessed 269 women with suspected or proven Lynch syndrome undergoing annual or biennial transvaginal ultrasound. The median age at the first investigation was 42 years. No cases of endometrial cancer were detected by surveillance during 826 patient years. However, two early-stage interval cancers did develop. Both cases were detected after a previous normal ultrasound, suggesting that annual or biennial ultrasound alone might not effectively detect early endometrial carcinoma.38 A retrospective study 39 of 41 women with known or suspected Lynch syndrome who had an annual transvaginal ultrasound included 197 patient-years, and ultrasonographic abnormalities were found in 17 of 179 visits. Subsequently, three premalignant lesions with complex atypical hyperplasia were found but no cases of endometrial cancer.39 One symptomatic interval cancer was found at an early stage 8 months after an ultrasound that detected no abnormalities.39 Renkonen-Sinisalo and colleagues 40 assessed gynaecological surveillance that consisted of transvaginal ultrasound and intrauterine biopsies of the endometrium. In 175 women with Lynch syndrome studied for 759 patient-years, 14 endometrial cancers were diagnosed, 11 as a result of surveillance. Only four endometrial cancers were detected by transvaginal ultrasound. Intrauterine sampling detected nine endometrial cancers and 14 premalignant hyperplasias. For patients with endometrial cancer, stage distribution and survival were more favourable in the surveilled than in the unsurveilled group. In the study by Renkonen-Sinisalo and colleagues,40 four patients were diagnosed with ovarian cancer that was previously undetected by transvaginal ultrasonography. 40 Surveillance for ovarian cancer is not eff ective in women with mutations in BRCA1 or BRCA2, which confer the highest risk for inherited ovarian cancer.41 Evidence does not support surveillance for ovarian cancer in patients with Lynch syndrome; however, data are scarce and studies on gynaecological surveillance have mainly focused on endometrial cancer. Surveillance for endometrial carcinoma and premalignant abnormalities is probably more eff ective with combined transvaginal ultrasound and endometrial biopsy than with ultrasound alone. Endometrial microsampling is a very accurate procedure that can be done in an office setting without anaesthesia.42 Further studies are needed to make recommendations on surveillance for ovarian cancer in women with Lynch syndrome.

Chemoprevention Women who take a combined oral contraceptive pill have a lower risk of endometrial and ovarian cancer than those who do not.43, 44 Furthermore, risk of these cancers decreases with increasing duration of oral contraceptive use. The protective effect on the ovaries is permanent whereas the protective effect on the endometrium seems to persist for at least 5 years after stopping of oral contraceptive use. Although evidence is insufficient to recommend oral contraceptives for the prevention of endometrial or ovarian cancer in women with Lynch syndrome, use is not contraindicated and might reduce risk of endometrial and ovarian cancers.

Prophylactic surgery Women with Lynch syndrome, especially those who need surgery for colon cancer, should be given the choice of prophylactic hysterectomy and bilateral salpingo-ophorectomy to eliminate the risk of endometrial and 186

RIBOGRAMA PROJECT ovarian cancer. Schmeler and co-workers 45 did a case–control study of 315 women with Lynch syndrome, one-third of whom had prophylactic hysterectomy and bilateral salpingo-oophorectomy. After a 10-year followup, endometrial cancer was detected in 33% and ovarian cancer in 5% of women in the control group, whereas no gynaecological cancers were diagnosed in the women who had prophylactic surgery.

Chen and colleagues 46 confi rmed the efficacy of prophylactic surgery for women with Lynch syndrome. They found that one diagnosis of endometrial cancer was prevented for every six surgeries and one diagnosis of ovarian cancer for every 28. Surgeons should be aware that undetected endometrial cancer can be present at the time of hysterectomy. Therefore, endometrial sampling before prophylactic surgery is recommended.47 If an occult endometrial cancer is found on preoperative investigations, surgeons should assess disease stage accordingly. No prospective studies have been done on symptoms, side-effects, and quality of life after prophylactic surgery in women with Lynch syndrome. In premenopausal women, prophylactic bilateral salpingo-oophorectomy results in premature menopause, and symptoms of hot flushes, decreased sexual interest, vaginal dryness, and disturbed sleep, but treatment of these symptoms with hormone replacement therapy is not contraindicated. Although evidence of eff ectiveness of gynaecological surveillance in women with Lynch syndrome is limited, current guidelines include annual transvaginal ultrasound and endometrial sampling starting at age 30–35 years.48, 49 Given the age distribution for development of endometrial cancer, we recommend surveillance from the age of 35–40 years. Women should be instructed to contact their physician in case of unusual signs or symptoms, such as unexpected vaginal bleeding. Prophylactic hysterectomy and bilateral salpingo-ophorectomy should be offered to women with Lynch syndrome who are past childbearing age, especially if surgery for colorectal cancer is needed. Hormone replacement therapy until the age of 50 years should be prescribed to all women with Lynch syndrome with premature surgical menopause.

Stomach cancer Epidemiology The lifetime risk of gastric cancer in people with Lynch syndrome varies substantially between populations. 4– 28 Lifetime risk is 2・1% in the Netherlands 8 and around 30% in Korea. 22 Clearly, risk is higher in areas that have high risk of gastric cancer in the general population, such as in Asia.22 Many patients with Lynch syndrome who develop gastric cancer are diagnosed before the age of 50 years, whereas 90% of sporadic gastric cancers are found after the age of 55 years.22 Gastric cancer seems to affect equal proportions of individuals with mutations in MLH1 and MSH2;16,50 although in Finland the risk was higher in carriers of mutations in MLH1 than MSH2.7 However, MLH1 mutations are the more common of the two in Finland.

Gastric cancer in individuals with Lynch syndrome is usually of the intestinal type and has a high degree of microsatellite instability.51 Intestinal-type gastric cancer is thought to develop through Helicobacter pylori infection (the most common proven risk factor),52 chronic atrophic gastritis, and intestinal metaplasia to dysplasia. Eradication of H pylori before any preneoplastic lesions are present, such as atrophy or intestinal metaplasia, could reduce the risk of gastric cancer.

Surveillance Currently, there is an increasing trend to recommend surveillance of the stomach by upper gastrointestinal endoscopy, although no consensus exists. Surveillance of the stomach by upper gastrointestinal endoscopy is recommended if more than one family member has gastric cancer.49 Clustering of gastric cancer within families, especially those with mutations in MSH2, has been suggested 13 but not confi rmed.50 Another proposal is to screen in countries with a high incidence of gastric cancer.49 Gastric surveillance was not 187

RIBOGRAMA PROJECT recommended, but could be off- ered periodically, in guidelines set out by Lindor and co-workers.48 A German group has proposed regular gastric surveillance in patients with Lynch syndrome beginning at age 35 years, although no age-specific rates were given to justify this recommendation. 50 No neoplastic lesions were found with gastroduodenoscopy in 73 patients with Lynch syndrome and a median age of 49 years.53 Although some investigators suggest regular gastroduodenoscopy for all individuals at risk, we think evidence is insufficient to justify such an approach. Surveillance should be considered in families with clustering of gastric cancer and in countries with a high prevalence. Additionally, we suggest H pylori detection and subsequent eradication in patients with Lynch syndrome.

Small-bowel cancer Epidemiology For people with Lynch syndrome, the lifetime risk of small bowel cancer is around 4%: over 100 times the risk in the general population.54 A review of available case series reported a median age at diagnosis of between 39 years and 53 years.54 Compared with the general population, patients with Lynch syndrome who develop small-bowel cancer usually present 10â&#x20AC;&#x201C;20 years earlier, and the risk is slightly higher in men than in women.54 Tumours are usually in the duodenum or the jejunum, with a small proportion in the ileum. The incidence of small-bowel cancer is similar in carriers of MLH1 and MSH2 mutations and somewhat rare in those with mutations in MSH6 and PMS2. At the molecular level, most adenocarcinomas show high levels of microsatellite instability.55 Surveillance Surveillance for small-bowel cancer is not in the guidelines for clinical management of families with Lynch syndrome because, until recently, visualisation of the small bowel was limited. Capsule endoscopy and double balloon enteroscopy has substantially improved accessibility of the small bowel. Small-bowel capsule endoscopy, in which patients swallow an optical capture device,56 is a safe, patient-friendly, minimally invasive technique for visualization of the small bowel. Although helpful in the diagnosis of small-bowel cancer,57 the technique is limited by the inability to obtain tissue for histological diagnosis or to provide endoscopic interventions. Double balloon enteroscopy is a reliable approach to detect lesions in the small bowel and allows direct access to the area.58 With capsule endoscopy as the most acceptable and potentially sensitive surveillance test for small bowel cancer, the usefulness and cost-eff ectiveness of surveillance with this technique warrants investigation. Until results are available from an ongoing study in Europe, physicians should discuss the risks and benefits of such a strategy with individual patients.

Pancreatic and biliary cancer Epidemiology Early single-family studies reported an association between Lynch syndrome and pancreatic or biliary cancer.

59â&#x20AC;&#x201C;61 The first larger series, done by Mecklin and co-workers,62 assessed 18 patients with pancreatic or biliary cancer from families with suspected Lynch syndrome. 15 patients had carcinoma of the biliary tract or papilla of Vater. Mean age at the time of diagnosis was 56 years.62 Geary and colleagues13 recently reported an incidence of pancreatic cancer seven times higher than in the general population in 319 individuals with Lynch syndrome. Most individuals with pancreatic cancer were diagnosed before the age of 60 years and 188

RIBOGRAMA PROJECT there was evidence of family clustering. Cancer of the biliary tract was rare.13 No data indicate a high risk of hepatocellular cancer in people with Lynch syndrome.

Surveillance Endoscopic ultrasound is the most sensitive technique for the detection of early pancreatic cancers 63 and studies of surveillance in individuals with Lynch syndrome are ongoing. So far, no surveillance approaches are successful in detecting pancreatic cancer at an early, possibly curable stage—not even in high risk groups, such as people with familial atypical multiple mole melanoma (FAMM) syndrome.64 Early detection of cancer of the biliary tract is even more difficult than pancreatic cancer. Surveillance for pancreatic or biliary cancer is not recommended for people with Lynch syndrome.

Urinary-tract cancer Epidemiology In patients with Lynch syndrome, the risk of urinary tract cancer, in particular cancer of the renal pelvis and ureter, is up to 12%.4,7,8,10–16,65 Watson and colleagues16 assessed 2683 proven or probable carriers of pathogenic MLH1 or MSH2 mutations and reported cumulative risks to the age of 70 years of: 6% (8% including bladder cancer risk) for the total group, 0.4 % (1%) for women with MLH1 mutations, 2% (4%) for men with MLH1 mutations, 9% (12%) for women with MSH2 mutations, and 20% (28%) for men with MSH2 mutations.16 The highest risks were seen between the ages of 50 and 70 years. No data are available on the risk of urinary tract cancer in people with PMS2 and MSH6 mutations.

Surveillance Ideally, upper urinary-tract cancer is detected in asymptomatic patients at an early stage when kidney-sparing therapy is possible. Detection of early stage bladder cancer would be an additional benefit. Very little is known about surveillance for urinary tract cancer in people with Lynch syndrome. The only report on urinary tract surveillance with urine cytology indicated a sensitivity of only 29% 66 probably a result of the low number of cancer cells discarded in the urine. Immunocytochemical testing, 67 or β-glucuronidase activity testing,68 improves the sensitivity of detection of upper urinary tract cancer, although sensitivity remains low. The fluorescence in-situ hybridization assay 69 might be highly sensitive but requires selective washing of the upper tract and is therefore classified as invasive. Testing for microsatellite instability in urine 70 to identify tumour DNA seems promising; however, data on its use in a surveillance setting are scarce. Of the possible imaging methods, abdominal ultrasound is not very sensitive for the detection of urinary-tract cancer.71 Urography with CT is more sensitive than ultrasound 7 but is not recommended because of the expense and repeated exposure to radiation. We believe that testing for haematuria with a simple and inexpensive urine dipstick is the most attractive approach to surveillance for urinary-tract cancer.72,73 Recommendations differ for the surveillance of urinary tract cancer in individuals with Lynch syndrome. Lindor and co-workers 48 advocate annual urinalysis with cytology, because of the low cost and non-invasiveness, without stratification of risk groups. Vasen and colleagues 49 recommend urinalysis with cytology and abdominal ultrasound, at an annual to biannual interval, beginning at the age of 30–35 years in families with a history of urinary tract cancer. Watson and colleagues16 propose surveillance from age 50 years, especially for families that carry mutations in MSH2. Watson’s group also recommend that preventive measures should not be limited to families with a history of urinary-tract cancer.16 We recommend annual surveillance for 189

RIBOGRAMA PROJECT haematuria by urine dipstick. If haematuria is confi rmed by urine microscopy, patients should have cystoscopy and abdominal ultrasonography—with a contrast study of the upper urinary tract in case of negative ultrasonography. We believe that surveillance should begin at age 45–50 years. In families with a history of urinary-tract cancer before age 45 years, surveillance should begin 5 years before the earliest age at diagnosis.

Pancreatic and biliary cancer Epidemiology Early single-family studies reported an association between Lynch syndrome and pancreatic or biliary cancer.

59–61 The first larger series, done by Mecklin and co-workers,62 assessed 18 patients with pancreatic or biliary cancer from families with suspected Lynch syndrome. 15 patients had carcinoma of the biliary tract or papilla of Vater. Mean age at the time of diagnosis was 56 years.62 Geary and colleagues13 recently reported an incidence of pancreatic cancer seven times higher than in the general population in 319 individuals with Lynch syndrome. Most individuals with pancreatic cancer were diagnosed before the age of 60 years and there was evidence of family clustering. Cancer of the biliary tract was rare.13 No data indicate a high risk of hepatocellular cancer in people with Lynch syndrome.

Surveillance Endoscopic ultrasound is the most sensitive technique for the detection of early pancreatic cancers63 and studies of surveillance in individuals with Lynch syndrome are ongoing. So far, no surveillance approaches are successful in detecting pancreatic cancer at an early, possibly curable stage—not even in high risk groups, such as people with familial atypical multiple mole melanoma (FAMM) syndrome.64 Early detection of cancer of the biliary tract is even more difficult than pancreatic cancer. Surveillance for pancreatic or biliary cancer is not recommended for people with Lynch syndrome.

Urinary-tract cancer Epidemiology In patients with Lynch syndrome, the risk of urinary tract cancer, in particular cancer of the renal pelvis and ureter, is up to 12%.4,7,8,10–16,65 Watson and colleagues16 assessed 2683 proven or probable carriers of pathogenic MLH1 or MSH2 mutations and reported cumulative risks to the age of 70 years of: 6% (8% including bladder cancer risk) for the total group, 0.4 % (1%) for women with MLH1 mutations, 2% (4%) for men with MLH1 mutations, 9% (12%) for women with MSH2 mutations, and 20% (28%) for men with MSH2 mutations.16 The highest risks were seen between the ages of 50 and 70 years. No data are available on the risk of urinary tract cancer in people with PMS2 and MSH6 mutations.

Surveillance Ideally, upper urinary-tract cancer is detected in asymptomatic patients at an early stage when kidney-sparing therapy is possible. Detection of early stage bladder cancer would be an additional benefit. Very little is known about surveillance for urinary tract cancer in people with Lynch syndrome. The only report on urinary tract surveillance with urine cytology indicated a sensitivity of only 29%:66 probably a result of the low number of 190

RIBOGRAMA PROJECT cancer cells discarded in the urine. Immunocytochemical testing, 67 or β-glucuronidase activity testing, 68 improves the sensitivity of detection of upper urinary tract cancer, although sensitivity remains low. The fluorescence in-situ hybridization assay 69 might be highly sensitive but requires selective washing of the upper tract and is therefore classified as invasive. Testing for microsatellite instability in urine 70 to identify tumour DNA seems promising; however, data on its use in a surveillance setting are scarce. Of the possible imaging methods, abdominal ultrasound is not very sensitive for the detection of urinary-tract cancer.71 Urography with CT is more sensitive than ultrasound 7 but is not recommended because of the expense and repeated exposure to radiation. We believe that testing for haematuria with a simple and inexpensive urine dipstick is the most attractive approach to surveillance for urinary-tract cancer.72, 73 Recommendations differ for the surveillance of urinary tract cancer in individuals with Lynch syndrome. Lindor and co-workers 48 advocate annual urinalysis with cytology, because of the low cost and non-invasiveness, without stratification of risk groups. Vasen and colleagues 49 recommend urinalysis with cytology and abdominal ultrasound, at an annual to biannual interval, beginning at the age of 30–35 years in families with a history of urinary tract cancer. Watson and colleagues 16 propose surveillance from age 50 years, especially for families that carry mutations in MSH2. Watson’s group also recommends that preventive measures should not be limited to families with a history of urinary-tract cancer.16 We recommend annual surveillance for haematuria by urine dipstick. If haematuria is confirmed by urine microscopy, patients should have cystoscopy and abdominal ultrasonography—with a contrast study of the upper urinary tract in case of negative ultrasonography. We believe that surveillance should begin at age 45–50 years. In families with a history of urinary-tract cancer before age 45 years, surveillance should begin 5 years before the earliest age at diagnosis.

Skin cancer Epidemiology Sebaceous tumours (adenoma, epithelioma, or carcinoma) and keratoacanthomas are prevalent in people with Lynch syndrome: an association known as Muir-Torre syndrome. South and colleagues 74 report MuirTorre syndrome in 14 of 50 families and 14 of 152 individuals with Lynch syndrome. Although predominantly associated with MSH2 mutations, Muir-Torre syndrome can also occur in families with mutations in MLH1 or MSH6.74,75 Skin tumours associated with Lynch syndrome often appear on the face, but can develop anywhere on the body. Most patients who develop skin lesions do so after diagnosis of another malignant disease associated with Lynch syndrome, but in some cases, skin cancer is the first sign of the syndrome. In a recent population-based study, patients with sebaceous carcinoma had a 43% increased risk of a second malignancy, especially in the colon, pancreas, and endometrium, compared with the general population.76 Individuals diagnosed with sebaceous carcinoma before the age of 50 years had a higher risk for subsequent cancers compared with individuals diagnosed over the age of 50 years.76 Surveillance No recommendations on surveillance for skin lesions among patients with Lynch syndrome are available.

Given the high rate of skin lesions, regular dermatological examination is a reasonable strategy for detection, especially in families with clustering of Muir-Torre syndrome. However, the age at which to start surveillance and the optimum interval are unclear. Patients who are at risk should be counselled to be alert for skin abnormalities and seek early medical attention.

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RIBOGRAMA PROJECT Brain tumours Epidemiology Lynch syndrome is associated with an increased risk of brain tumours.7, 8, 11, 14–16, 77 In 6041 proven or probable carriers of pathogenic MLH1 or MSH2 mutations, or their first-degree relatives, cumulative risk of brain tumours to the age of 70 years was 2% for the total group, 1,7% for carriers of MLH1 mutations, and 2・5% for carriers of MSH2 mutations.16 Risks might have been underestimated because individuals who developed brain tumours as children were less likely to be identified. Median age at the time of brain tumour diagnosis is lower in those with Lynch syndrome than in the general population; 26% of the diagnoses were made before the age of 25 years.16 The most common tumour types are glioblastoma multiforme and astrocytoma, but oligodendrogliomas and ependymomas have also been reported. Brain tumours in individuals with Lynch syndrome are rarely associated with microsatellite instability.78 Despite a low incidence, brain tumours were the third highest cancer-related cause of death in a large Dutch cohort of patients with Lynch syndrome. 3 Surveillance No studies have investigated surveillance for brain tumours in Lynch syndrome. Given the low risk of brain tumours associated with this syndrome and the absence of surveillance methods known to decrease morbidity and mortality, surveillance has not been recommended.

Other tumours Other tumour types that have been reported in patients with Lynch syndrome include breast, prostate, larynx, lung, thyroid, and testicular cancers, and melanoma, lymphoma, leukaemia, and soft tissue sarcomas.16,79– 83 Molecular findings showed that mismatch repair deficiency contributed to the development of those tumours.79–83 Evidence does not show an increased risk of breast or prostate cancer for individuals with Lynch syndrome, although breast cancer risk is somewhat controversial and might differ between populations.84,85 The absolute risk for the other cancer types listed is probably too low to justify surveillance, even if effective methods are available. Ponti and co-workers 79 suggest surveillance for melanomas already identified in families with Lynch syndrome but the effectiveness of this strategy is unknown. In families suspected to have Lynch syndrome but without the more typical colorectal or endometrial cancers, a practical approach might be to study molecular signs of mismatch repair deficiency in selected rare tumour types in the diagnostic work-up.

Conclusion In this review, we discussed management of extracolonic malignancies in Lynch syndrome with an emphasis on surveillance. For individuals with Lynch syndrome, the colorectum is the only organ in which surveillance reduces the risk of cancer and mortality. We advocate discussing with the patient the risks and surveillance strategies for other types of tumours associated with Lynch syndrome. Nilbert and colleagues 86 confirmed the need to start surveillance at a young age by showing genetic anticipation—earlier age at onset of cancer in successive generations.

The guidelines and recommendations for surveillance of extracolonic malignancies in people with Lynch syndrome are not supported by solid evidence. Although the risk of extracolonic tumours might be higher in 192

RIBOGRAMA PROJECT individuals with mutations in MSH2 compared with MLH1, 5,8,12 and the risks in MSH6 and PMS2 mutation carriers are not well known, no evidence indicates that surveillance strategies should differ. We recommend that surveillance starts at age 40â&#x20AC;&#x201C;45 years with annual urinanalysis dipstick test and that surveillance is not limited to families with a history of urinary-tract tumours. For gastric cancer, we propose once only serological H pylori determination and eradication. The outcome of each surveillance strategy for patients with Lynch syndrome should be systematically collected and analysed.

Data are limited with respect to chemoprevention to reduce cancer risk in Lynch syndrome. A recent randomised placebo-controlled trial studied the efficacy of aspirin and resistant starch on reducing the risk of colorectal neoplasia in Lynch syndrome.87 The use of either treatment, alone or in combination, for up to 4 years, had no effect on the incidence of colorectal neoplasia.

No data show efficacy of chemopreventive agents in reducing the risk of extra-colonic neoplasia. We have focused on management of patients who are proven carriers of mutations associated with Lynch syndrome. In practice, these recommendations are also applied to first-degree relatives of known mutation carriers who decline genetic testing, and patients and their first-degree relatives from families suspected to have Lynch syndrome but with unknown mutations. Surveillance should be preceded by genetic counselling about the natural history of the syndrome and the need for long-term follow-up. Counselling should acknowledge that the recommendations are generally based on expert opinions rather than evidence from clinical trials.

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68. Ho KJ, Kuo SH. Urinary beta-glucuronidase activity as an initial surveillance test for urinary tract malignancy in high risk patients. Comparison with conventional urine cytologic evaluation. Cancer 1995; 76: 473–78 69. Chen AA, Grasso M. Is there a role for FISH in the management and surveillance of patients with upper tract transitional-cell carcinoma? J Endourol 2008; 22: 1371–74.

70. Ho CL, Tzai TS, Chen JC, et al. The molecular signature for urothelial carcinoma of the upper urinary tract. J Urol 2008; 179: 1155–59.

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78. Gylling AH, Nieminen TT, Abdel-Rahman WM, et al. Differential cancer predisposition in Lynch syndrome: insights from molecular analysis of brain and urinary tract tumors. Carcinogenesis 2008; 29: 1351–5.

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XXV - CLINICAL AND LABORATORY APPLICATIONS OF AMNIOTIC MEMBRANE AND FLUID IN STEM CELL BIOLOGY A. Ferreira-Alemao, MD PhD UNIVERSIDAD COMPLUTENSE DE MADRID

1. Introduction The amniotic membrane (AM) and amniotic fluid (AF) have a long history of use in surgical and prenatal diagnostic applications, respectively. In addition, the discovery of cell populations in AM and AF which are widely accessible, nontumorigenic and capable of differentiating into a variety of cell types has stimulated a flurry of research aimed at characterizing the cells and evaluating their potential utility in regenerative medicine. While a major focus of research has been the use of amniotic membrane and fluid in tissue engineering and cell replacement, AM- and AF-derived cells may also have capabilities in protecting and stimulating the repair of injured tissues via paracrine actions, and acting as vectors for biodelivery of exogenous factors to treat injury and diseases. Much progress has been made since the discovery of AM and AF cells with stem cell characteristics nearly a decade ago, but there remain a number of problematic issues stemming from the inherent heterogeneity of these cells as well as inconsistencies in isolation and culturing methods which must be addressed to advance the field towards the development of cell-based therapies. Here, we provide an overview of the recent progress and future perspectives in the use of AM- and AF-derived cells for therapeutic applications.

Regenerative medicine involves the use of living cells to repair, replace, or restore normal function to damaged or defective tissues and organs (1, 2). Stem cells are viewed as promising candidates for use in cell-based therapies, owing to their capacity for self-renewal and differentiation into diverse mature progeny. However, the source of stem cells, in order to maximize the safety and efficacy of regenerative therapies, is clearly of great importance. Both adult and embryonic stem cells are commonly used to develop therapies for various preclinical models of disease and injury. Recently, induced pluripotent stem (iPS) cells, which are obtained by genetically reprogramming adult somatic cells to a pluripotent state, have also been proposed as an alternative cell source for use in regenerative medicine (3, 4). However, a number of limitations hamper the clinical applicability of stem cells derived from either adults or developing embryos. While embryonic stem cells (ES cells) are highly proliferative and capable of differentiating into cells of all adult tissues, they pose a significant risk of tumour formation (5). Furthermore, since ES cells are obtained by the destruction of embryos, they face serious ethical objections that have yet to be resolved. In contrast, although adult stem cells carry a reduced risk of tumorigenicity and fewer ethical restrictions, they are limited in number, have diminished differentiation capacity, and reduced proliferative potential (6, 7) which render the production of a sufficient number of cells for use in cell-based therapy difficult. Finally, despite major advances in iPS technology in recent years, reprogrammed cells often have an imperfectly cleared epigenetic memory of the source cells (8). In addition, iPS cells are vulnerable to genomic instability 199

RIBOGRAMA PROJECT (9, 10). Due to the drawbacks associated with ES cells, adult stem cells and iPS cells, much effort has been directed at finding an alternative source of cells for use in regenerative medicine.

Subpopulations of multipotent cells exist in both the amniotic membrane (AM) and amniotic fluid (AF).

Amniotic fluid cells are obtained during amniocentesis, an important diagnostic procedure performed worldwide to evaluate the health status of the fetus during pregnancy. Amniotic epithelial (AE) and amniotic mesenchymal stromal (AMS) cells are isolated from amnion that is normally discarded following birth. These cells are therefore readily available, easily procured, and avoid the ethical issues that are associated with the use of ES cells. Subpopulations of AF- and AM-derived cells with stem cell characteristics can be maintained in the undifferentiated state in culture, but are capable of differentiating into cells representing all three germ layers under appropriate conditions (11,12). Unlike ES cells, AF and AE cells have not been found to form teratomas when transplanted in vivo (11, 1316) and may be a safer alternative to ES cells. A comparison of AF, AE and AMS stem cells with ES cells is provided in Table 1 The use of amniotic fluid- and membrane-derived cells as cell-based therapy for a variety of indications has been extensively explored in the past decade. Here, we briefly review the findings regarding the use of AM and AF in tissue engineering and cell replacement strategies in a number of injury and disease models.

Table 1: Comparison of ES, AM and AF stem cells. â&#x20AC;&#x2030;

Embryonic stem cells

Amniotic epithelial cells

Source

Inner cell mass of preimplantation embryo 300+ population doublings (48)

Amniotic membrane 14 population doublings (49)

Ectodermal, mesodermal, endodermal (53) Yes (54) Yes Yes (55)

Ectodermal, mesodermal, endodermal (20) No (15) No Yes (56)

In vitro lifespan Differentiation potential in vitro Tumorigenicity Ethical issues Clinical trials

Amniotic mesenchymal stromal cells Amniotic membrane

Amniotic fluid cells

5â&#x20AC;&#x201C;10 passages(50), 27 population doublings (51) Ectodermal, mesodermal, endodermal (20) Not known No No

55 (52) to 250+ (14) population doublings Ectodermal, mesodermal, endodermal (14) No (14) No No

Amniotic fluid

Table 1: Comparison of ES, AM and AF stem cells.

2. Amniotic Membrane 2.1. Amniotic Membrane Is a Natural Scaffold with Multiple Clinical Applications Human amniotic membrane is the innermost fetal layer, lining the amniotic cavity and protecting the fetus during pregnancy. The outer layer, termed chorionic membrane, further separates the fetus from maternal tissues. Reports focusing on the physiological functions of fetal layers have shown that amniotic membrane not only provides a physical support for the fetus, but also serves as a metabolically active filter through a direct interaction with amniotic fluid. In particular, the transport of water and soluble materials as well as the production of growth factors, cytokines, and other bioactive 200

RIBOGRAMA PROJECT molecules are regulated by amniotic membrane (17). In addition to its role during pregnancy, amniotic membrane allows the initiation and maintenance of uterus contraction at birth (18).

The isolation of human fetal membranes from the placenta. (a) Note the texture and elasticity of the membranes. (b) Human amniotic (left) and chorionic (right) membranes can be readily separated from each other for further purification procedures.The translucent, avascular, low immunogenic, antiinflammatory, antiscarring, and wound healing properties of amniotic membrane allow this material function beyond its role in vivo and assume a wide range of applications in regenerative medicine (19, 20). In fact, the clinical use of amniotic membrane has a long history, with the first reports on its application in treatment of skin burns and wounds more than a century ago (21-23). These groundbreaking studies played a significant role in advancing the use of amniotic membrane in surgery, especially in areas such as reconstruction of the corneal and conjuctival surfaces, treatment of open ulcers and traumatic wounds, and skin transplantation (17,20,24,25). In parallel, the shelf life of amniotic membrane has been extended by irradiation, air-drying, lyophilization, cryo-preservation, and glycerol preservation techniques. These methods are expected to further expand the use of amniotic membrane in ophthalmology to treat corneal, conjunctival and limbal lesions, burns, scars and defects as well as general surgery to reconstruct skin, genitourinary tract and other surfaces (25-31).

However, the efficacy of amniotic membrane in clinical applications can only be enhanced by retaining its biological properties in the long term. This issue is especially important because the presence of key growth factors such as EGF, FGF, TGF, and HGF in amniotic membranes may account for their clinical effects and mechanisms of action. Currently, a series of standardized guidelines are being developed in a number of countries to optimize the production of surgically suitable amniotic membrane from donor placenta.

2.2. Stem Cells in Amniotic Membrane In addition to these strategies, various histological, biochemical, and cellular biology techniques have been used to isolate and determine the suitability of the cells in amniotic membrane for other clinical applications. Epithelial cells can be readily identified as a single layer adjacent to the amniotic fluid on one side and the basement membrane on the other side (17,32,33). While epithelial cells reside on the inner layer of the amniotic membrane, mesenchymal stromal cells form the outer layer (17,32,33).

Both cell types have been extensively investigated for their biological properties, using a number of in vitro and in vivo models. In particular, the expression of several cellular and molecular markers has confirmed the presence of stem cells in epithelial and mesenchymal stromal cultures. Subpopulations of both AE cells and AMS cells express pluripotency markers, including OCT4, SOX2, and NANOG (13,15,34,35). AE cells express embryonic stem cell markers such as SSEA4, TRA-1-60, and TRA-181 (13,36) while reports on the expression of ES cell markers by AMS cells have been inconsistent (20). Technical issues have prevented researchers from determining whether a single human AE or AMS (hAE or hAMS) cell can differentiate into cells representative of all three germ layers after clonal expansion (37); therefore, it remains unclear whether the human amnion harbours true pluripotent stem cells, or a mixed population of multipotent progenitor cells. Nevertheless, it is widely accepted that multiple cell types can be derived by culturing either AE or AMS cells under appropriate conditions. Several laboratories have reported neural (13,15,35,38,39), hepatic (13,15,40-43), cardiac (15,34,44), osteogenic (15,45,46) chondrogenic (39,47) and adipogenic (15,46) differentiation of both AE and AMS cells.

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RIBOGRAMA PROJECT 2.3. Amniotic Membrane-Derived Cells in Tissue Engineering and Cell Replacement The development of biological substitutes to replace damaged or dysfunctional tissue may involve the construction of “replacement parts” in vitro for later transplantation, or the direct administration of cells to the damaged tissue (57) AE and AMS cells have been employed for both purposes. For instance, after inducing osteogenic differentiation of human AMS cells seeded onto microcarriers, the resulting bone-like structures could be used as building blocks to form a large (2 × 1 cm) bone construct in vitro (58). AE cells have been used to form tendon-like structures (59), and a double-layered skin graft (using both AE and AMS cells) capable of repairing skin defects in mice (60). Human AE and AMS cells have also been shown to reduce liver damage in a chemically-induced model of cirrhosis (61,62) and improve cardiac function after experimental cardiac infarction (34,44,63). Furthermore, both AE and AMS cells were able to replace insulin-producing pancreatic beta cells in diabetic mice to restore normal glucose levels (64-66). Comprehensive reviews of the differentiation potential and therapeutic use of AE and AMS cells in experimental models are available in the literature (18,20,37,67- 69).

3. Amniotic Fluid 3.1. Amniotic Fluid Is a Dynamic Environment Containing Diverse Cell Types Human amniotic fluid is a dynamic environment, which undergoes multiple developmental changes in order to sustain fetal growth and well being (Figure 2). The amniotic cavity first appears at 7-8 days after fertilization and in early gestation the amniotic fluid originates mostly from maternal plasma that crosses the fetal membranes (70). Fetal urine first enters the amniotic space at 8–11 weeks gestation (70), and in the second half of pregnancy, fetal urine becomes the major contributor to amniotic fluid (71). At this time, fetal skin keratinisation is complete, leading to reduced water transport across the skin and a decrease in AF osmolality. For the remainder of gestation, fluid volume is determined by different mechanisms including fetal urine production, oral, nasal, tracheal and pulmonary fluid secretion, fetal swallowing, and the contributions of the intramembranous pathway (72).

The fluid is mostly derived from maternal plasma at this gestational age. A 2D ultrasound image of the fetus at 20 weeks (c) and a 3D ultrasound image of the fetal head at 36 weeks. Fetal urine is the main contributor to the fluid at this gestational age. Note the difference in proportion of amniotic fluid in the first (a) and second (c) trimesters.

Amniotic fluid contains electrolytes, growth factors, carbohydrates, lipids, proteins, amino acids, lactate, pyruvate, enzymes, and hormones (73-76). In addition, fluid secretions from the fetus into the AF carry a variety of fetal cells, resulting in a heterogeneous population of cells derived from fetal skin, gastrointestinal, respiratory and urinary tracts, and the amniotic membrane (77). As the fetus develops, the volume and composition of the amniotic fluid change drastically, and the complement of cells detected in amniotic fluid samples taken at different gestational ages varies considerably (78,79).

Despite this heterogeneity, cultures of amniotic fluid cells obtained by amniocentesis have been used for decades for diagnostic purposes, including standard karyotyping as well as other genetic and molecular tests. AF samples are routinely used in the evaluation of fetal lung maturity, metabolic diseases, fetal infections, and intrauterine infections. These tests have recently been complemented by applying chromosomal microarray (CMA) as a more efficient prenatal genetic screening tool to detect fetal abnormalities (80). In this multicenter study, nearly 4400 AF samples were used to assess the performance of CMA compared with karyotyping for prenatal cytogenetic diagnosis. Interestingly, 202

RIBOGRAMA PROJECT CMA analysis allowed the detection of additional genetic abnormalities in about 1 out of every 70 samples that reported a normal karyotype during routine prenatal diagnosis. These results further emphasize the importance of AF cells in providing clinically important information about the fetus. In addition, this technology can be used to routinely follow the status of different subpopulations of amniotic fluid cells in culture and identify the most suitable clones for cell-based therapies.

Generation and banking of monoclonal human AF stem cell lines with specific chromosomal aberrations or monogenic disease mutations may also help study the functional consequences of disease-causing mutations (81,82). As a promising approach, the use of prolonged siRNA-mediated gene silencing in AF stem cells (83) may advance our understanding of the functions of specific genes and shed light on the pathogenesis of certain naturally occurring diseases (84).

3.2. Stem Cells in Amniotic Fluid The fact that amniotic fluid is commonly collected for routine diagnostic testing and is a widely accessible source of fetal cells, prompted an interest in examining the possibility that AF might contain multipotent fetal-derived cells (85). In 2003, Prusa et al. discovered the existence of a small population of actively dividing cells in human amniotic fluid which express OCT4, a marker of pluripotent stem cells, as well as stem cell factor, vimentin and alkaline phosphatase (86). In the same year, In â&#x20AC;&#x2122;t Anker et al. reported the isolation of mesenchymal stem cells with multilineage differentiation capacity from amniotic fluid (87). A subsequent study used immunoselection for c-kit (CD117, receptor for stem cell factor) to isolate a population of cells with high self-renewal capacity that expressed some common ES cell markers (OCT4 and SSEA4) as well as markers of somatic stem cells (CD29, CD44, CD73, CD90, and CD105) that are not typically detected in ES cells (14).

Several AF-derived clonal cell lines were established that exhibited the capability to differentiate into cell types from all three germ layers (including adipogenic, osteogenic, myogenic, endothelial, neurogenic, and hepatic cells) (14). A number of other studies have also investigated the differentiation capacity of clonal AF-derived cells (88-93). However, evaluation of the differentiation potential of AF-derived cells has relied heavily on expression of selected markers. Thus, further research is required to demonstrate that differentiated cells are capable of acquiring functional characteristics of the desired cell type, especially in vivo.

3.3. AF Cells in Tissue Engineering and Cell Replacement Because they are readily accessible, pose little to no ethical concerns, and do not form teratomas in vivo, amniotic fluid-derived cells have been investigated as a promising alternative source of cells for use in tissue engineering and cell-based therapies. Kaviani et al. first demonstrated that mesenchymal cells from ovine or human AF could be seeded on synthetic scaffolds, as a prelude to using these cells for tissue engineering (94,95). Since that time, amniotic fluid-derived cells have been used in experimental settings to repair different tissues, including cartilage grafts for fetal tracheal reconstruction (96), tendons for diaphragm repair (97,98) bone grafts (99-101), and heart valve leaflet (102-104). In vivo administration of amniotic fluid-derived cells as a strategy for cell replacement has had beneficial effects in various injury models, including acute bladder injury (105), acute tubular necrosis of the kidney (106), hyperoxic lung injury (107) and ischemic heart (108). The use of AF cells in tissue engineering and cell replacement has been extensively reviewed elsewhere (11,12,20) and is summarized in Table 2.

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RIBOGRAMA PROJECT Table 2: Applications of AF stem cells.

AF cell source

Target tissue

Human

Brain

Human Human

Brain Brain

Normal and twitcher neonatal mice Mouse cerebral ischemia Rat cerebral ischemia

Rat

Brain

Rat cerebral ischemia

Human

Brain

Mouse motor cortex injury

Human

Nerve

Rat sciatic nerve crush injury

Human

Nerve, Muscle

Rat sciatic nerve crush injury

Human

Heart

Rat cardiac infarction

Rat

Heart

Human

Lung, Heart

Rat cardiac infarction Rat pulmonary hypertension and heart failure

Sheep

Heart valve

Mouse

Delivery route

References

Intracerebroventricular injection

(14)

Intracerebroventricular injection Intrastriatal injection Intravenous injection into the jugular vein Injection or implantation of cells seeded on biocompatible scaffolds into the motor cortex Injection or implantation of cells and fibrin glue into the injury site Intravenous injection Intracardiac injection of cells or cell sheet fragments Intracardiac injection

(109) (110)

(108,121,122)

Intravenous injection into the tail vein

(123)

Fetal sheep

Closed-heart implantation of cells seeded on biodegradable scaffolds in utero

(104)

Skeletal muscle

Mouse spinal muscular atrophy

Intravenous injection into the tail vein

(124)

Human

Bone

Mouse subcutaneous implantation

Rabbit

Bone

Rabbit chest wall/sternal defects

Human

Bone

Rat subcutaneous implantation

Sheep

Cartilage

Sheep

Diaphragm

Human

Kidney

Rat Rat

Bladder Abdomen Fetal membranes

Fetal lamb tracheal reconstruction Postnatal sheep diaphragmatic hernia Mouse kidney acute tubular necrosis Rat cryo-injured bladder Rat Fetal rabbit iatrogenic membrane defect

Subcutaneous implantation of cells printed on biocompatible polymers Bone graft implantation of cells seeded on biocompatible scaffolds into the injury site Subcutaneous implantation of cells seeded on biocompatible polymers Tracheal implantation of cells seeded on biocompatible scaffolds in utero Diaphragmatic implantation of cells seeded on biocompatible scaffolds

Sheep

Nonspecific

Fetal lamb organs

Mouse, Human

Hematopoietic

Mouse

Rabbit

Animal/disease model

(111) (112) (113-117) (118) (119,120)

(14) (99) (101) (96) (97)

Injection into the renal cortex

(106)

Intravascular injection Intraperitoneal injection Injection into the plug followed by fixation to the fetal membranes Injection into the fetal peritoneal cavity in utero Intravenous injection into the retroorbital vein

(105) (125) (126) (127) (128)

Table 2: Applications of AF stem cells.

4. Complementary Applications of AE, AMS, and AF Cells 4.1. Paracrine Action of AF- and AM-Derived Cells in Tissue Repair A common theme among several studies attempting to use AF, AE, or AMS cells for tissue repair in injury models is that, despite improving organ function, these cells often do not differentiate into the desired cell type or integrate fully into the target tissues (105,129). This issue may be particularly 204

RIBOGRAMA PROJECT pertinent in neural applications, since the ability of AF-derived stem cells to differentiate into neurons has been a matter of debate (130,131) and definitive evidence that AF, AE, or AMS stem cells can be induced to become mature functional neurons in vivo is still lacking. Nevertheless, the use of amniotic membrane- and fluid-derived cells for nervous system repair has met with some success. c-kit+ AF cells injected into injured chick embryo spinal cord increased embryo survival and reduced injuryinduced haemorrhaging, although the cells failed to undergo terminal differentiation into neurons (132). Pan et al. (113,114) reported that AF-derived mesenchymal stromal cells improved motor function and electrophysiological indicators of nerve function in a sciatic nerve crush model, in the absence of stem cell penetration into the nerve. AF cells have also been shown to improve memory and sensory/motor functions following focal ischemia induced by middle cerebral artery occlusion (MCAO) in mice as soon as 4 days after cell injection (109). Although the fate of the injected cells was not examined in that study, it is doubtful that AF cells could have differentiated into mature neurons capable of effectively integrating into the host circuitry to restore function on such a short time scale.

Therefore, it is unlikely that cell replacement could directly account for the beneficial effects of AF cells in this study. In a rat model of Parkinsonâ&#x20AC;&#x2122;s disease, implantation of AE cells into rat striatum prevented the degeneration of nigrostriatal dopaminergic neurons, when administered prior to the neurotoxin 6OHDA (133), and attenuated motor disturbances in rats that had previously been subjected to 6OHDA-induced degeneration (134). Subsequent work showed that administration of AE cells into the lateral ventricle had a similar effect, which was maintained over 10 weeks despite the fact that the majority of the transplanted cells either did not survive, or did not exhibit a dopaminergic phenotype at the end of the experiment (135). These results further suggest that the positive effect of the transplanted AE cells was not due to their ability to replace lost nigrostriatal neurons.

In a number of cases, the favourable outcomes observed after AF or AM cell transplants have been attributed not to the direct replacement of lost cells, but rather to factors secreted by the cells which may serve a protective or reparative function. Such paracrine mechanisms have also been postulated to explain some of the positive effects of other stem cell types in animal models of organ/tissue injury (136-138). Studies in which conditioned media (CM), rather than AF or AM cells themselves, have been used in injured tissues support the notion that secreted factors mediate, at least in part, the beneficial effects of the transplanted cells. For instance, AF-CM (139) and AMS-CM (140) both reinstated blood flow in a murine hindlimb ischemia model, and AF-CM increased perfusion to an ischemic skin flap (141) likely owing to the presence of proangiogenic growth factors and cytokines, including VEGF, SDF-1, and TGF-Ă&#x; present within the media. AF-CM was also shown to stimulate other endogenous repair mechanisms, such as proliferation of dermal fibroblasts near the injury site in a mouse excision wound model (142) and recruitment of endothelial progenitor cells to ischemic skin flap (141). Other paracrine mechanisms, such as the production of trophic factors (114,143) immunomodulation (144,145), and creation of a supportive milieu for regeneration (146) might also contribute to the ability of AF- or AM-derived cells to limit damage and/or stimulate repair of injured tissue.

4.2. AF- and AM-Derived Cells for Delivery of Beneficial Factors Although AF- and AM-derived cells appear to have natural protective and reparative functions, they may also be used for efficient biodelivery of specific factors to enhance the protection or repair of damaged tissue through genetic modification. Accordingly, it was recently reported that AF mesenchymal stromal cells engineered to express elevated levels of GDNF ameliorated motor deficits in rats subjected to sciatic nerve crush, beyond the improvement observed with green fluorescent protein (GFP)-transduced cells (147). To extend this research to CNS applications, we are currently 205

RIBOGRAMA PROJECT assessing the neuroprotective capacity of GDNF-expressing AF cells in a mouse motor cortex injury model (unpublished data). Both AE (148) and AMS (149) cells have also been used to deliver neurotrophic factors (GDNF and BDNF, resp.) to ischemic rat brain, and in both cases, enhanced recovery using GDNF- or BDNF-expressing cells was observed, relative to GFP-expressing cells.

AF- and AM-derived cells might be suitable for delivery of diverse compounds for a variety of diseases. For instance, a handful of recent studies have made use of AF cells for biodelivery of anticancer therapeutics. Yin et al. engineered AF mesenchymal stromal cells to express the antiangiogenic factor endostatin and the prodrug-activating enzyme secretable carboxylesterase 2 (sCE2) to treat glioma. sCE2 converts the antitumour drug CPT11 into its active form. By injecting the engineered cells along with glioma-forming cells prior to treatment with CPT11, the AF cells boosted the conversion of the prodrug to its active form selectively at the tumour site, inhibiting proliferation, increasing apoptosis, and decreasing the population of glioma stem cells (150). Similarly, expression in AF cells of cytosine deaminase and thymidine kinase, which act as suicide genes by converting two cancer prodrugs to their active toxic forms, inhibited the growth of breast tumours in a xenograft mouse model and prevented both the damage to the surrounding tissue and the physical side effects that were observed when the active drugs were directly administered (151). These studies highlight a potential role for AF cells in biodelivery of a wide range of compounds.

Presumably, all of the above-mentioned studies have relied on bulk release of secreted factors into the extracellular space to mediate the beneficial effects of AF or AM cells. However, we are also investigating the possibility that AF cells could be used for direct cellular delivery of certain types of molecules via gap junctional communication, as has been suggested by Brink et al. (152) for bone marrow mesenchymal stromal cells. AF cells express connexins, the proteins that make up gap junction hemichannels, and are capable of establishing gap junctional communication with cultured cortical cells, as evidenced by dye transfer (112). Given the induction of connexin expression surrounding a surgical lesion to motor cortex, (112) as well as in other models of brain injury (153155), it is hoped that AF cells might be capable of delivering small molecules through gap junctions to host cells, in an effort to protect the surrounding tissue or promote repair mechanisms.

5. Current Limitations in the Use of AM and AF Cells Recent evidence suggests that diverse subpopulations of multipotent cells in amniotic fluid differ in marker expression, morphology, and/or growth kinetics (16,156). Furthermore, amniotic membranederived cells are not as homogeneous as previously thought. Different culture conditions and methods for isolating and expanding cells with stem cell characteristics might introduce a bias towards producing particular subpopulations of cells (11). In addition, the gestational stage at which AF is collected (79) and the passage number of the cultured cells (157) will likely influence the resulting cell phenotypes and behaviour. At present, it is not clear exactly what effects these methodological differences have on the outcome of studies, but there is an agreement that cells used by different research groups may not represent identical biological properties. While this renders the comparison of different studies very difficult, it also prompts the question of whether different subpopulations of multipotent cells in AF and AM have distinct differentiation capacities. There is, in fact, some evidence that this is the case (156,158,159). Further exploration of this issue is required, and hopefully it will be possible to exploit these differences to isolate cells with greater potential to differentiate into desired functional cell types. This should be done in conjunction with an examination of the role of culture conditions in directing AF, AE, and AMS cell differentiation towards particular cell fates.

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Furthermore, it is possible that predifferentiation of AF- or AM-derived cells toward a desired phenotype prior to transplantation might promote engraftment in some tissues (160,161). This issue warrants further investigation, especially considering the low rate of differentiation of transplanted AMor AF-derived cells observed in many studies.

Finally, although AM and AF-derived cells reportedly possess low immunogenicity and can survive transplantation into xenogeneic or allogeneic hosts (14,20,61,62,146,162), one study found that AF cells were rejected upon transplantation into immunocompetent animals due to the recruitment of host T and B lymphocytes, natural killer cells and macrophages (163). In another case, poor survival of amniotic epithelium grafts was observed in mice that received repeated transplants, because of immune rejection (164). Other studies have also reported a low rate of survival of transplanted AF cells (114,165,166), which may be a result of immune rejection. Thus, as for ES cells, whose status as immune-privileged has been questioned (167), further research is required in order to understand the immunological properties of AM- and AF-derived cells, and to enhance graft survival.

There is a need for the establishment of national and international registries of cell lines derived from amniotic membrane and fluid in order to make these lines available to researchers worldwide. This strategy will facilitate the development of guidelines for the derivation and characterization of new cell lines and provide detailed protocols for culturing and differentiating existing lines. It is expected that the proposed approach would reduce methodological variabilities, which are compounded by the inherent heterogeneity of amniotic cells. In addition, the creation of a library of information pertaining to the research and (pre)clinical use of AF, AE, and AMS cells would allow researchers to choose the most appropriate cell line for a particular application, hopefully leading to more rapid development of effective regenerative therapies.

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RIBOGRAMA PROJECT 120. Y. C. Yeh, H. J. Wei, W. Y. Lee et al., “Cellular cardiomyoplasty with human amniotic fluid stem cells: in vitro and in vivo studies,” Tissue Engineering—Part A, vol. 16, no. 6, pp. 1925–1936, 2010.

121. W. Y. Lee, H. J. Wei, W. W. Lin et al., “Enhancement of cell retention and functional benefits in myocardial infarction using human amniotic-fluid stem-cell bodies enriched with endogenous ECM,” Biomaterials, vol. 32, no. 24, pp. 5558–5567, 2011.

122. L. Iop, A. Chiavegato, A. Callegari et al., “Different cardiovascular potential of adult- and fetal-type mesenchymal stem cells in a rat model of heart cryoinjury,” Cell Transplantation, vol. 17, no. 6, pp.

679–694, 2008.

123. A. Angelini, C. Castellani, B. Ravara et al., “Stem-cell therapy in an experimental model of pulmonary hypertension and right heart failure: role of paracrine and neurohormonal milieu in the remodeling process,” Journal of Heart and Lung Transplantation, vol. 30, no. 11, pp. 1281–1293, 2011.

124. M. Piccoli, C. Franzin, E. Bertin et al., “Amniotic fluid stem cells restore the muscle cell niche in a HSA-Cre, SmnF7/F7 mouse model,” Stem Cells, vol. 30, no. 8, pp. 1675–1684, 2012.

125. M. Ghionzoli, M. Cananzi, A. Zani et al., “Amniotic fluid stem cell migration after intraperitoneal injection in pup rats: implication for therapy,” Pediatric Surgery International, vol. 26, no. 1, pp. 79– 84, 2010.

126. D. Liekens, L. Lewi, J. Jani et al., “Enrichment of collagen plugs with platelets and amniotic fluid cells increases cell proliferation in sealed iatrogenic membrane defects in the foetal rabbit model, ”Prenatal Diagnosis, vol. 28, no. 6, pp. 503–507, 2008.

127. S. W. Steven Shaw, S. Bollini, K. A. Nader et al., “Autologous transplantation of amniotic fluidderived mesenchymal stem cells into sheep fetuses,” Cell Transplantation, vol. 20, no. 7, pp. 1015– 1031, 2011.

128. A. Ditadi, P. De Coppi, O. Picone et al., “Human and murine amniotic fluid c-Kit+Lin- cells display hematopoietic activity,” Blood, vol. 113, no. 17, pp. 3953–3960, 2009.

129. C. Rota, B. Imberti, M. Pozzobon et al., “Human amniotic fluid stem cell preconditioning improves their regenerative potential,” Stem Cells and Development, vol. 21, no. 11, pp. 1911–1923, 2012.

130. M. Toselli, E. Cerbai, F. Rossi, and E. Cattaneo, “Do amniotic fluid-derived stem cells differentiate into neurons in vitro?” Nature Biotechnology, vol. 26, no. 3, pp. 269–270, 2008.

131. M. Rosner, M. Mikula, A. Preitschopf, M. Feichtinger, K. Schipany, and M. Hengstschläger, “Neurogenic differentiation of amniotic fluid stem cells,” Amino Acids, vol. 42, pp. 1591–1596, 2011.

132. W. Prasongchean, M. Bagni, C. Calzarossa, P. De Coppi, and P. Ferretti, “Amniotic fluid stem cells increase embryo survival following injury,” Stem Cells and Development, vol. 21, no. 5, pp. 675– 688, 2012 133. K. Kakishita, N. Nakao, N. Sakuragawa, and T. Itakura, “Implantation of human amniotic epithelial cells prevents the degeneration of nigral dopamine neurons in rats with 6-hydroxydopamine lesions,” Brain Research, vol. 980, no. 1, pp. 48–56, 2003.

134. K. Kakishita, M. A. Elwan, N. Nakao, T. Itakura, and N. Sakuragawa, “Human amniotic epithelial cells produce dopamine and survive after implantation into the striatum of a rat model of Parkinson's disease: a potential source of donor for transplantation therapy,” Experimental Neurology, vol. 165, no. 1, pp. 27–34, 2000.

135. X. X. Yang, S. R. Xue, W. L. Dong, and Y. Kong, “Therapeutic effect of human amniotic epithelial cell transplantation into the lateral ventricle of hemiparkinsonian rats,” Chinese Medical Journal, vol. 122, no. 20, pp. 2449–2454, 2009.

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RIBOGRAMA PROJECT 136. N. Joyce, G. Annett, L. Wirthlin, S. Olson, G. Bauer, and J. A. Nolta, “Mesenchymal stem cells for the treatment of neurodegenerative disease,” Regenerative Medicine, vol. 5, no. 6, pp. 933–946, 2010 137. D. De Feo, A. Merlini, C. Laterza, and G. Martino, “Neural stem cell transplantation in central nervous system disorders: from cell replacement to neuroprotection,” Current Opinion in Neurology, vol. 25, no. 3, pp. 322–333, 2012.

138. W. Dai, G. L. Kay, A. J. Jyrala, and R. A. Kloner, “Experience from experimental cell transplantation therapy of myocardial infarction: what have we learned?” Cell Transplantation. In press.

139. M. Teodelinda, C. Michele, C. Sebastiano, C. Ranieri, and G. Chiara, “Amniotic liquid derived stem cells as reservoir of secreted angiogenic factors capable of stimulating neo-arteriogenesis in an ischemic model,” Biomaterials, vol. 32, no. 15, pp. 3689–3699, 2011.

140. H. G. Kim and O. H. Choi, “Neovascularization in a mouse model via stem cells derived from human fetal amniotic membranes,” Heart and Vessels, vol. 26, no. 2, pp. 196–205, 2011.

141. T. Mirabella, J. Hartinger, C. Lorandi, C. Gentili, M. Van Griensven, and R. Cancedda, “Proangiogenic soluble factors from amniotic fluid stem cells mediate the recruitment of endothelial progenitors in a model of ischemic fasciocutaneous flap,” Stem Cells and Development, vol. 21, no.

12, pp. 2179–2188, 2012.

142. B. S. Yoon, J. H. Moon, E. K. Jun et al., “Secretory profiles and wound healing effects of human amniotic fluid-derived mesenchymal stem cells,” Stem Cells and Development, vol. 19, no. 6, pp.

887–902, 2010.

143. S. Uchida, Y. Inanaga, M. Kobayashi, S. Hurukawa, M. Araie, and N. Sakuragawa, “Neurotrophic function of conditioned medium from human amniotic epithelial cells,” Journal of Neuroscience Research, vol. 62, no. 4, pp. 585–590, 2000.

144. K. Kamiya, M. Wang, S. Uchida et al., “Topical application of culture supernatant from human amniotic epithelial cells suppresses inflammatory reactions in cornea,” Experimental Eye Research, vol. 80, no. 5, pp. 671–679, 2005.

145. E. C. Moorefield, E. E. McKee, L. Solchaga et al., “Cloned, CD117 selected human amniotic fluid stem cells are capable of modulating the immune response,” PLoS ONE, vol. 6, no. 10, Article ID e26535, 2011.

146. V. Sankar and R. Muthusamy, “Role of human amniotic epithelial cell transplantation in spinal cord injury repair research,” Neuroscience, vol. 118, no. 1, pp. 11–17, 2003.

147. F. C. Cheng, M. H. Tai, M. L. Sheu et al., “Enhancement of regeneration with glia cell line-derived neurotrophic factor-transduced human amniotic fluid mesenchymal stem cells after sciatic nerve crush injury,” Journal of Neurosurgery, vol. 112, no. 4, pp. 868–879, 2010.

148. T. Liu, J. Wu, Q. Huang et al., “Human amniotic epithelial cells ameliorate behavioral dysfunction and reduce infarct size in the rat middle cerebral artery occlusion model,” Shock, vol. 29, no. 5, pp.

603–611, 2008.

149. J. Tao, F. Ji, B. Liu, F. Wang, F. Dong, and Y. Zhu, “Improvement of deficits by transplantation of lentiviral vector-modified human amniotic mesenchymal cells after cerebral ischemia in rats, ”Brain Research, vol. 1448, pp. 1–10, 2012.

150. J. Yin, J. K. Kim, J. H. Moon et al., “HMSC-mediated concurrent delivery of endostatin and carboxylesterase to mouse xenografts suppresses glioma initiation and recurrence,” Molecular Therapy, vol. 19, no. 6, pp. 1161–1169, 2011.

151. N.-H. Kang, K.-A. Hwang, B.-R. Yi et al., “Human amniotic fluid-derived stem cells expressing cytosine deaminase and thymidine kinase inhibits the growth of breast cancer cells in cellular and xenograft mouse models,” Cancer Gene Therapy, vol. 19, no. 6, pp. 412–419, 2012.

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RIBOGRAMA PROJECT 152. P. R. Brink, V. Valiunas, C. Gordon, M. R. Rosen, and I. S. Cohen, “Can gap junctions deliver?”Biochimica et Biophysica Acta, vol. 1818, no. 8, pp. 2076–2081, 2012.

153. N. Rouach, E. Avignone, W. Même et al., “Gap junctions and connexin expression in the normal and pathological central nervous system,” Biology of the Cell, vol. 94, no. 7-8, pp. 457–475, 2002.

154. A. Ohsumi, H. Nawashiro, N. Otani, H. Ooigawa, T. Toyooka, and K. Shima, “Temporal and spatial profile of phosphorylated connexin43 after traumatic brain injury in rats,” Journal of Neurotrauma, vol. 27, no. 7, pp. 1255–1263, 2010.

155. C. Haupt, O. W. Witte, and C. Frahm, “Up-regulation of Connexin43 in the glial scar following photothrombotic ischemic injury,” Molecular and Cellular Neuroscience, vol. 35, no. 1, pp. 89–99, 2007.

156. M. G. Roubelakis, V. Bitsika, D. Zagoura et al., “In vitro and in vivo properties of distinct populations of amniotic fluid mesenchymal progenitor cells,” Journal of Cellular and Molecular Medicine, vol. 15, no. 9, pp. 1896–1913, 2011.

157. Y. W. Kim, H. J. Kim, S. M. Bae et al., “Time-course transcriptional profiling of human amniotic fluid-derived stem cells using microarray,” Cancer Treatment and Research, vol. 42, pp. 82–94, 2010.

158. S. Arnhold, S. Glüer, K. Hartmann et al., “Amniotic-fluid stem cells: growth dynamics and differentiation potential after a CD-117-based selection procedure,” Stem Cells International, vol.

2011, Article ID 715341, 12 pages, 2011.

159. J. Bai, Y. Wang, L. Liu et al., “Human amniotic fluid-derived c-kit+ and c-kit- stem cells: growth characteristics and some differentiation potential capacities comparison,” Cytotechnology. In press.

160. X. Wang, Y. Lu, H. Zhang et al., “Distinct efficacy of pre-differentiated versus intact fetal mesencephalon-derived human neural progenitor cells in alleviating rat model of Parkinson's disease,” International Journal of Developmental Neuroscience, vol. 22, no. 4, pp. 175–183, 2004.

161. H. Aurich, M. Sgodda, P. Kaltwaßer et al., “Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo,” Gut, vol. 58, no. 4, pp.

570–581, 2009.

162. C. A. Akle, M. Adinolfi, and K. I. Welsh, “Immunogenicity of human amniotic epithelial cells after transplantation into volunteers,” The Lancet, vol. 2, no. 8254, pp. 1003–1005, 1981.

163. A. Chiavegato, S. Bollini, M. Pozzobon et al., “Human amniotic fluid-derived stem cells are rejected after transplantation in the myocardium of normal, ischemic, immuno-suppressed or immunodeficient rat,” Journal of Molecular and Cellular Cardiology, vol. 42, no. 4, pp. 746–759, 2007.

164. M. Wang, A. Yoshida, H. Kawashima, M. Ishizaki, H. Takahashi, and J. Hori, “Immunogenicity and antigenicity of allogeneic amniotic epithelial transplants grafted to the cornea, conjunctiva, and anterior chamber,” Investigative Ophthalmology and Visual Science, vol. 47, no. 4, pp. 1522–1532, 2006.

165. A. E. Donaldson, J. Cai, M. Yang, and L. Iacovitti, “Human amniotic fluid stem cells do not differentiate into dopamine neurons in vitro or after transplantation in vivo,” Stem Cells and Development, vol. 18, no. 7, pp. 1003–1011, 2009.

166. R. Soler, C. Fllhase, A. Hanson, L. Campeau, C. Santos, and K.-E. Andersson, “Stem cell therapy ameliorates bladder dysfunction in an animal model of Parkinson disease,” Journal of Urology, vol.

187, no. 4, pp. 1491–1497, 2012.

167. R. J. Swijnenburg, S. Schrepfer, F. Cao et al., “In vivo imaging of embryonic stem cells reveals patterns of survival and immune rejection following transplantation,” Stem Cells and Development, vol. 17, no. 6, pp. 1023–1029, 2008.

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XXVI - PRESERVATION SOLUTION FOR COLONOCYTES A. Ferreira-Alemao, MD PhD UNIVERSIDAD COMPLUTENSE DE MADRID

The object of the present saline solution is to provide a transport medium and a dispersion or suspension medium for isolating viable exfoliated fecal colonocytes, isolated at normal ambient temperature in accordance with the teachings of the present description.

The method comprises the steps of: a) collecting a fecal sample in a transport medium at normal ambient temperature; b) dispersing the fecal sample in said transport medium diluted with a suspension medium; c) sedimenting cells present in the diluted transport medium of step b) to isolate the cells from impurities by layering the cell suspension over a medium of heavier density; d) centrifuging the cells in step C) to form a cellular band at a boundary with said heavier medium and within the heavier medium and pellet, thereby obtaining isolated intact viable exfoliated colonocytes from said fecal sample.

Description The present method is related to isolated, biologically substantially pure and viable colonocytes obtained from a fecal sample. It provides a transport medium and a dispersion or suspension medium for isolating viable colonocytes from a fecal sample at normal ambient temperature employing the isolated colonocytes.

Colonocytes represent an important source of informational marker molecules that provide a picture of the immediate past metabolic history of the GI tract of a subject. Such cells are representative of the cell population from a statistically large sampling frame reflecting the state of the colonic mucosa along the entire length of the colon in a non-invasive manner, in contrast to a limited sampling by colonic biopsy using an invasive procedure involving endoscopy.

Colonocytes undergo certain changes or transformations and carry certain biological or chemical markers indicative of colonic pathologies including precancerous and cancerous conditions. The colonocytes could serve as a valuable early indicator of the onset of neoplastic processes and other pathophysiological changes in the GI tract. It is generally understood that exfoliated colonic cells are destroyed once they are shed into the stools, the reason being that these cells start breaking down as soon as they are exposed to the atmosphere.

The enzymes, mucus and the bacteria contained in the stool contribute to the process of destroying the colonic cells.

Dutta and Nair (Gastroenterology, 114:1333-1335, 1998) refer to Albaugh et al (Int. J. Cancer, 52:347-350, 1992) and Iyengar et al (FASEB J. 5: 2856-2859, 1991) for accomplishing isolation of viable colonic cells.

Albaugh et al described a transport medium and a procedure to obtain colonocytes from a stool sample based on earlier work of Iyengar et al. However, the transport medium of the prior art was different from the transport medium of the present method in as much as Albaugh et al's medium consisted of a saline solution to which antibiotics were added in addition to fatty acid free BSA. The prior art transport medium was deficient at least in one criterion, i.e., in not having a mucolytic agent, which is an absolute necessity for the formulation of the transport medium in accordance with the present invention.

218

RIBOGRAMA PROJECT Furthermore, in Albaugh et al's system the stool sample after collection had to be kept cooled in ice while being transported to the laboratory for further processing and could be preserved in ice only for about an hour.

In contrast, the system of the present invention does not require cooling in ice and achieves desirable results at the normal ambient temperature which is an important feature of the present invention. Moreover, Albaugh et al state that although their cells had viability in excess of 80%, the presence of phagocytes and other cells could not be ruled out. Thus, the procedures of Iyengar et al and Albaugh et al may provide viable colonocytes, but they are inadequate for the purpose of obtaining substantially pure colonocytes. In summary, heretofore it has not been possible to obtain a viable, biologically substantially pure sample of a particular cell type isolated at normal ambient atmospheric conditions from a small fecal mass.

Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are only exemplary and not limiting. The term "substantially pure" as used herein means that the product is homogeneous or uniformly of a single type with greater than 96% purity, usually 98%-99% pure, as determined by flow cytometry or by the procedures described herein and is without material interference or contamination of other types of cells.

The problem encountered with exfoliated colonocytes in fecal matter was that these cells got destroyed as soon as exposed to the normal atmospheric conditions due to the presence in the fecal matter of proteolytic enzymes, microflora, mucus and the like. A system is devised to inhibit the action of all those factors or elements, which prevented the isolation of intact, living colonocytes at normal ambient temperature from fecal matter.

This is achieved by the present method by formulating two different media: (1) a transport medium, and (2) a dispersion or suspension medium. The transport medium is made of physiological saline solution containing an enzyme-inactivating amount of an enzyme trapping or protease sequestering agent, a sufficient amount of a bactericidal agent to inhibit bacterial activity, and a mucolytic amount of an agent that destroys mucus contained in fecal matter.

Various enzyme trapping, bacteriostatic and mucolytic agents will be suggested to one of ordinary skill in the art and any suitable agents that do not interfere with the objects of the present method could be used in the formulation of the transport medium in accordance with the teachings of the present description. Preferred among enzyme trapping agents are proteolytic activity inhibitors and animal proteins. Examples of suitable proteolytic activity inhibitors include PMSF, pepstatin A, bestatin and chymostatin. The reagents that inhibit or deactivate enzymatic activity include formaldehyde, metal-chelating agents, heavy metal ions, certain amino acids such as tyrosine and phenylalanine, and high concentrations of zinc or inorganic phosphates.

Suitable among animal proteins are those that are non-immune, water-soluble compounds including serum albumins of rabbit (RSA), goat (GSA), sheep (SHA), horse (ESA), bovine (BSA) and human (HSA) origin.

Certain polyamino acids that do not interfere with a later assay procedure could also be used as enzyme deactivating agents. Suitable examples of such polyamino acids are poly-L-lysine, poly-L-proline, poly-ltyrosine and the like. Suitable examples of bacteriostatic agents are sodium azide, sodium benzoate, antibiotics (such as penicillin, streptomycin, amphotericin B, gentamicin, polymyxin B and the Like). A preferred bacteriocidal agent is Thimerosal (Sigma Chemical Co.) Suitable examples of mucolytic agents are guaifenesin, guaiacol, potassium iodide, B-mercaptoethanol, dithiothreitol, capsaicin, glycyrrhizin and the like.

A preferred mucolytic agent is N-Acetyl Cysteine (Sigma Chemical Co.).

Puck's Saline G is a preferred source of physiological saline solution.

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RIBOGRAMA PROJECT A preferred transport medium is prepared as follows: Puck's Saline G.......................500 ml Sodium Bicarbonate.................350 to 500 mgs BSA ......................................2.5 to 15 mgs N-Acetyl Cysteine...................250 to 500 mgs Thimerosal.............................100 to 300 mgs The dispersion or suspension medium differs from the transport medium with respect to bactericidal agent, e.g., Thimerosal, which is omitted when preparing the dispersion or suspension medium.

Procedure for Isolating Substantially Pure Colonocytes at Normal Ambient Temperature The stool sample is placed into a container containing the transport medium, after which the container is closed with a stopper. The stool sample is then thoroughly dispersed in medium and diluted with the dispersion medium and mixed in a mechanical mixer (e.g., Stomacher, Tekmar Co.) for about 30 seconds. The contents of the container are then allowed to settle down briefly. The aliquot of the container is then pipetted into 50 ml polypropylene conical centrifuge tubes and underlaid with a heavy medium having a density in the range of about 1.033 to 1.120 (preferably Histopaque 1119, Sigma Chemical Co.) and centrifuged at about 200.times.g for about 30 minutes in a table top centrifuge with the brakes off.

The colonic cells accumulate as a band at the interface between the heavy medium and the lighter suspension above. This band comprising the desired colonic cells is removed by sucking out with a plastic transfer pipette and placed in a new 50 ml centrifuge tube and diluted with about 40 ml of the dispersion medium. The suspension of cells thus obtained is then centrifuged at about 900.times.g for about 10 minutes after which the clear supernatant is discarded and the cell pellet remaining at the bottom of the centrifuge tube is resuspended in phosphate buffered saline (PBS) containing about 1% bovine serum albumin (BSA). The suspension is then again centrifuged at 900.times.g after which the clear supernatant is discarded and the pellet, comprising substantially biologically pure isolated exfoliated viable colonocytes, is recovered. For determining the viability of the isolated colonocytes, a portion of the pellet is dispersed in PBS/BSA medium (1 ml/gm of stool sample).

Then, a 1/10 dilution of the suspension is counted in a hemocytometer in the presence of trypan blue. As is well known to a skilled artisan, the cells that do not take up trypan blue are considered to be viable and counted to determine the cell yield.

It is important to note that an inventive aspect of the present method is the formulation of a transport medium and a dispersion or suspension medium which together allows the exfoliated colonocytes found in the fecal sample to remain viable at the normal ambient temperature during and after the isolation procedure.

The method enables isolation of intact, living exfoliated colonocytes from a small sample (e.g. 3 gm) of fecal material without chilling or freezing, at normal ambient temperature ranging from about 22 degree C. to about 24 degree C. during the entire isolation process. About 8 to 10 million living colonic cells can be obtained from one gram of the stool sample in accordance with the techniques of the present method.

When maintained at the normal ambient temperature in the suspension medium of the present invention, the isolated colonocytes can be viably preserved for several hours. Table 1 shows cell yields and viability as a function of storage time and temperature conditions. Alternate techniques could be substituted in the isolation steps. For example, the suspension of the stool sample in the transport medium may be filtered through screens (149 micrometers, 105 micrometers and 52 micrometers). Also, the pellet in the dispersion medium can be gently overlayered on higher density Percoll gradients and centrifuged so that the cells can be recovered from the top of the gradient. Such modifications are common in the art and are included within the purview of this invention.

220

RIBOGRAMA PROJECT It is understood, of course, that whenever appropriate a reference or base line would be usually established using colonocytes obtained from disease-free subjects so that a comparative, diagnostic or evaluation study could be made with colonocytes obtained from a subject suspected of a disease or pathological condition. It was discovered that an important and advantageous feature of the non-invasively obtained colonocytes of the present method is that these isolated colonic cells carry markers or transformations characteristic of the pathology of the GI tract.

As a source of somatic cells obtainable non-invasively, the colonocytes of the present method are representative of the phenotype as well as genotype. These isolated cells are also useful in various other ways easily suggested to a skilled artisan. It should be apparent that given the guidance, illustrations and examples provided herein, various alternate embodiments, modifications or manipulations of the present method would be suggested to a skilled artisan and these are included within the spirit and purview of this application and scope of the appended claims.

TABLE 1 - Effect of storage on cell yields and viability Storage Time (Hrs)

Conditions

Cell Yield (%)

Viability (%)

1-6

Ambient

100

85+

10

Ambient

100

65

24

4 degree C

40

50

36

4 degree C

24

5-6

References 1. Albaugh et al, Int. J. Cancer, 1992, 52:347-350.

2. Battistelli et al., Bollettino--Societa Italiana Biologica Sperimentale, 1990, 66:985-992.

3. Dorland's Illustrated Medical Dictionary, 26th Ed., 1985, W.B. Saunders Company, Philadelphia, p.

342

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XXVII - SUPPORT PROTOTYPE OF FAECES COLLECTION (COLLECTOR) SUMMARY OF COLECTOR PARTS

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COLLECTOR PARTS BREAKDOWN Figure. 1

Figure. 2

Figures 1-2 Description: Support standard collector/container-top-exterior, which will apply in the toilet bowl in the toilet seat being compressed (fixed and immobile) when the patient-user feel for their needs. This support has a socket where the upper rim of the container is immobile, as seen in Figures 1-2.

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Figure. 3

Figure. 4

Container/higher external collector Figures 3-4 Description: Container/collector-interior of feces, which is embedded in the part of Figures 1-2, in which side is the container/collector-filtering indoor grill like a piston as if it were a syringe, as shown in Figures 5-6. The container/collector-interior of Figures 3-4 has a central hole in the bottom, like a cylindrical funnel that will fit in a container/collector below (Figures 5-6), where he will collect a washing (serum appropriate for maintaining viability of cells in the mucosa colo rectal/colonic). This stool is washing the immersion of the stool in the serum (whose chemical composition is explained in elsewhere), which will flow to the container/collector-bottom as shown in Figures 7-8, after the valve depicted in Figures 9-10, re-opened by a valve mechanism itself, driven by pressure container/collective-exterior, coaxial with the container/collectorto-bottom. Also verify the existence of a reserve for closing airtight device, described in Figures 9-10.

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RIBOGRAMA PROJECT Figure 5

Figure 6

Container/lower collector

Figures 5-6 Description: Container/collector-less, looking cylindrical with an opening in the upper face, with the cylindrical funnel through which to fit, like a syringe in the other funnel, inside, so coaxial with the funnel at the top, described in Figures 3-4. The CSE, as the cylinder is adjusted lower stage on the upper stage of the container/collector-less

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RIBOGRAMA PROJECT Figure 7

Figure 8

Figures 7-8 Description: Cover for sealing of the container/catcher-upper-outer.

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RIBOGRAMA PROJECT Figure 9

Figure 10

Figures 9-10 Description: Hermitical closure device from the top of the container/catcher-upper-outer (that which contains a socket for functional closure device as can be seen in Figure 9-10).

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Figure 11

Figure 12

Figures 11-12 Description: Valve that allows the transit unidirectional container/catcher-upper-exterior to the container/collector-less.

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RIBOGRAMA PROJECT Figure 13

Figure 14

Figures 13-14 Description: Tri-dimensional Vision (3D) of all stool collector.

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XXVIII â&#x20AC;&#x201C; TREATMENT COSTS OF BOWEL CANCER IN ENGLAND Bowel cancer is a leading cause of mortality and morbidity in England. It accounts for 12% of all cancers in England. In 2003, there were approximately 27,800 new cases registered in England, making it the third most common cancer after breast and lung cancer. The number of deaths resulting from bowel cancer in 2003 in the UK was 7,488 for men and 6,466 for women, resulting in an estimated 154,182 premature life years lost.

Historically, survival rates in England are lower than those in many European and North American countries.

Five year survival rates for patients in England and Wales with bowel cancer are 42% for men and 40% for women (for those diagnosed between 1991-93). This compares with a five year survival rate for Europe of around 50% for women and between 45 - 49% for men. The recent Eurocare study indicated that compared to Europe, patients in the United Kingdom present at a more advanced stage in the disease and that a lower percentage of patients are resected.

BACKGROUND TO THE RESEARCH The Policy Research Programme of the Department of Health commissioned this research to identify the costs, activity and outcomes of current bowel cancer treatments and consider options for service reconfiguration. The study has been undertaken by a research team from the York Health Economics Consortium at the University of York and the School of Health and Related Research at the University of Sheffield. They have been supported by an Advisory Group established by the Department of Health, and external advisers.

OUTPUTS Two reports have been produced from this research study. This report quantifies the an activity, costs and outcomes, associated with the current treatment of bowel cancer and predicts the costs and outcomes of options for re-configuration of bowel cancer services. An accompanying report summarizes the literature reviewed as part of this the study.

METHODOLOGY Phase one of the study comprised developing and populating the treatment pathway for bowel cancer to reflect current practice. The pathway is based on evidence derived from published literature supplemented by expert opinion. The pathway was populated with data synthesised from a number of sources including published literature, expert opinion and audits. Phase two comprised developing a model to predict the impact of changes in the delivery of bowel cancer services on costs and outcomes, with a view to prioritizing options for service improvement.

THE TREATMENT PATHWAY The treatment pathway considers the following aspects of care: 230

RIBOGRAMA PROJECT • Pathway A – Access to bowel cancer services; • Pathway B – Treatment of colon cancer; • Pathway C – Treatment of rectal cancer; • Pathway D – Follow-up; • Pathway E – Surveillance of individuals with adenomatous polyps; • Pathway F – Management of increased-risk groups.

ASSESSMENT OF CURRENT COSTS, ACTIVITY AND OUTCOMES OF BOWEL CANCER DIAGNOSIS & TREATMENT The research estimates the total diagnosis and treatment costs of bowel cancer based on current service provision. Total annual diagnosis and treatment costs in England are estimated to be approximately £1.1billion. The largest component of the total cost is the cost of diagnosis which makes up 26.4%. The next most substantial cost is the follow-up cost of patients with bowel cancer which accounts for 24.7% of the total cost of illness. The mean cost per patient for rectal cancer treatment was estimated at £12,037 in comparison with the mean cost of colon cancer treatment of £8,808. The additional cost of screening by Faecal Occult Blood Testing for those aged 60 to 69 biennially was estimated to be £112.8m in the first full year, although these are predicted to fall in subsequent years. A summary of the cost of the main care components, aggregated for both colon and rectal cancers, is presented in the table below.

CARE COMPONENT TOTAL COST (£) Screening Cost (additional year 1 cost.) ............................................

Non-cancer patients (i.e. diagnosis costs to exclude cancer) ...........

Diagnosis costs for confirmed cancer patients ..................................

Primary Treatment (surgery, CT/RT) ..................................................

Follow-up (surveillance) ......................................................................

Recurrence ..........................................................................................

Stoma ...................................................................................................

Palliative ............................................................................................... (of which End of life care costs) ..........................................................

High-risk patients – surveillance .........................................................

Total Cost of Illness ...............

£112,828,886 £270,129,193 £ 20,595,291 £200,628,632 £ 24,402,914 £246,653,295 £ 52,076,567 £118,552,980 £ 21,408,066 £ 53,758,184 £1,099,625,942

OPTIONS ASSESSMENT Options considered in the model are summarized below: 1. Improving GP referral criteria for suspected colorectal pathology; 2. Raised bowel cancer awareness in the general population of England; 3. Management of emergency presentation and delivery of care; 4. Increasing the use of colonoscopy from 70 per cent to 90 per cent; 5. Improving colonoscopy completion rates via national training; 6. The use of laparoscopic versus open surgery for colon cancer patients; 7. Assessment of the impact of surgical expertise for outcomes and/or developing specialist pathology services; 8. Pre-operative versus post-operative radiotherapy for rectal cancer patients; 231

RIBOGRAMA PROJECT 9. The use of alternative chemotherapy sequencing: a. The use of alternative adjuvant chemotherapies; b. The use of alternative palliative sequences for chemotherapy.

10. The use of the Enhanced Recovery Programme (ERP) following surgery; 11. Intensive versus relaxed follow-up; 12. Increasing liver/lung resections for metastatic disease; 13. Increasing palliative surgery (e.g. palliative bypass) and stenting.

The options are intended to provide an indication of the potential cost effectiveness of each of the interventions described above. Prior to implementing any of the interventions, further research on their cost effectiveness, particularly taking into account a more detailed assessment of their costs, is recommended. The model suggests that increasing the use of colonoscopy from 70% to 90% is cost saving and would improve health outcomes. Future research concerning the natural history of the disease and the probability of polyps being detected through flexible sigmoidoscopy in patients with distal colon cancer would be valuable. The introduction of an Enhanced Recovery Programme is also cost saving with initial indications of a low associated risk of detrimental clinical outcomes. This option is again relatively robust and at an advanced stage of development for implementation. The model suggests that the most costly options would be the further development of GP referral criteria guidelines, although this is also associated with a reasonable improvement in life years gained. However, this option is highly uncertain due to the lack of evidence surrounding disease progression and the cost of implementation. It would also require substantial further research in order to identify referral criteria that will affect improve sensitivity without detrimental impact upon specificity.

Greater knowledge is required regarding the relationship between symptomology and disease progression.

Increasing the use of emergency stenting is expected to be very effective for a small number of patients consuming colorectal cancer resources, but is associated with a relatively high cost. The options to improve surgical expertise and/or pathology are associated with improvements in health outcomes and may be potentially cost saving. Similarly, the options to improve adjuvant or palliative chemotherapies are expected to improve health outcomes, although cost impact is more uncertain. The remainder of the options which were assessed had smaller effects on both costs and health outcomes. Many of the options assessed within the model display huge variability due to the large amount of uncertainty associated with both the base case model and the options. There is very little evidence regarding health utility scores for bowel cancer services, which makes it difficult to differentiate between the effectiveness of many of the options. This is a clear area in which further research would be merited.

SUMMARY This study is believed to represent the most robust attempt to capture the full costs of treating bowel cancer in England. The research estimates that bowel cancer costs almost ÂŁ1.1bn per annum to manage. The options appraisal exercise suggests that outcomes could be improved and in some cases costs reduced, through changes to the current treatment pathways.

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BACKGROUND AND METHODS RATIONALE FOR THE RESEARCH Bowel cancer, also known as colorectal cancer, causes a substantial number of deaths in England each year.

Historically, survival rates for patients with bowel cancer in England are lower than those in many countries of Europe and North America. Against the backdrop of the desire by the Government to improve cancer services, as laid out in The National Health Service (NHS) Cancer Plan of September 20001, the Policy Research Programme of the Department of Health has initiated a study to estimate the costs and benefits of bowel cancer services in England. In particular, the Department of Health wishes to examine how to allocate future investment in bowel cancer services to deliver optimal benefit to patients at an acceptable economic cost. This research study has been undertaken in response to the Department of Health objectives and aims to identify expenditure at a national level in England on bowel cancer services as a whole, as well as expenditure on the different elements of service provision. The study also seeks to quantify the likely costs and benefits of different options for the development of bowel cancer services, including the likely time scale for the costs and benefits to take effect.

SCOPE OF THE RESEARCH STUDY The Department of Health identified two distinct phases of research: • The first phase was to investigate the overall activity and expenditure on bowel cancer within the NHS (including affiliated organizations, such as hospices) as well as the patient outcomes that result from this activity; • The second phase was to investigate what improvements in outcomes might be achievable at what cost and within what timescale.

THE DEPARTMENT OF HEALTH HAS ALSO SPECIFIED A NUMBER OF AREAS THAT SHOULD BE ADDRESSED BY THE RESEARCH INCLUDING: • Earlier presentation and improved assessment in primary care; • Optimizing diagnostic services; • Optimizing potentially curative treatments; • Optimizing palliative treatment and care.

DEPARTMENT OF HEALTH SEPTEMBER (2000) “THE NHS CANCER PLAN: A PLAN FOR INVESTMENT, A PLAN FOR REFORM” The outputs of this study are intended to assist ministers and policy makers in the optimal allocation of health care resources. The research is intended to help identify areas in which current expenditure levels are inconsistent with the clinical and cost-effectiveness evidence base. The research is also intended to inform decisions about the re-allocation of resources to optimize the outputs of bowel cancer treatment. Finally, it is hoped that the methodology might provide a template to facilitate comparisons of expenditure at a network level and internationally. This research study has been undertaken through a collaboration between York 233

RIBOGRAMA PROJECT Health Economics Consortium (YHEC) at the University of York, and the Health Economics and Decision Science Group from the School of Health and Related Research at the University of Sheffield.

STRUCTURE OF THE REPORT This report comprises 6 sections: • Section 1 provides an introduction to the research study, some background Information about bowel cancer and its impact, and an overview of the methodology adopted for the research study; • Section 2 describes the pathways for the diagnosis, treatment and follow-up of individuals with bowel cancer (including those pathways followed by individuals without bowel cancer who consume bowel cancer service resources); • Section 3 describes the detail of the methodology and calculation of the costs, activity and outcomes for the current management of patients with bowel cancer in England; • Section 4 describes the methodology used to simulate the expected costs and health outcomes resulting from an identified set of potential options for service reconfiguration; • Section 5 describes the expected health outcomes and costs associated with each of the options for change; • Section 6 summarizes the research results including discussion and recommendations for further research.

BACKGROUND TO BOWEL CANCER IN ENGLAND Bowel cancer includes cancerous growths in the colon, rectum and appendix (cancer of the appendix has not been considered within the current study). The cancer cells may spread to nearby lymph nodes (local recurrences) and also to more remote lymph nodes and other parts of the body (metastatic recurrences). The liver and the lungs are common sites for metastatic spread. The most common symptoms on presentation are blood on or mixed with stools; change in bowel habit; anaemia; weight loss, nausea and anorexia; and abdominal pain. However, these symptoms are not exclusively associated with bowel cancer and are associated with a variety of benign conditions which are prevalent in the general population. Importantly, some symptoms may not become apparent until the cancer is at an advanced stage, by which time the prognosis is poor. Patients are likely to develop a variety of physical and psychological symptoms during the life of the disease. Seymour et al. (1997).

GUIDANCE ON SERVICE PROVISION Guidance was initially issued in 1997, and subsequently updated in 2000, on Improving Outcomes in Colorectal Cancer (IOCC 2004). The IOCC guidance document summarized the evidence to date on the management of those with bowel cancer and provided recommendations for best clinical practice. The evidence focuses on diagnosis, treatment and follow-up of patients. Options for the management of patients after diagnosis include combinations of surgery, radiotherapy and chemotherapy and palliative care for those patients who have a poor prognosis. The manual was not designed to provide a set of mandatory instructions or clinical practice guidelines. However, as stated in the manual: “By focusing on components of provision which are most relevant to patient outcomes, considering the resource implications of change, and by giving information on anticipated health benefits of implementing the 234

RIBOGRAMA PROJECT recommendations, the manual will help commissioners concentrate on areas most likely to make a difference” (NICE IOCC, 2004).

The National Institute for Health and Clinical Excellence (NICE) has issued guidance on: • • • • •

Referral of patients by GPs for all cancers, which includes bowel cancer; The use of laparoscopic and open surgery for bowel cancer; The use of adjuvant chemotherapy for colon cancer; The use of chemotherapy for patients with advanced and metastatic bowel cancer.

The care and treatment of patients with bowel cancer has been estimated to account for approximately 2% of all bed days and for between 10 - 20% of all palliative care provision in the UK. Mountney et al. (1994).

In 2003, the rates of newly diagnosed cases of bowel cancer for England were 62.3 for men and 49.5 for women per 100,000 population. The probability of developing bowel cancer increases sharply with age. In individuals below the age of 40 years, the risk is very low, with an incidence rate of 4.9 per 100,000 population in men and 4.0 per 100,000 population for women. However, between the ages of 40-49 years, the incidence rate rises to 20.9 per 100,000 population in men and 16.5 per 100,000 population in women. This increases further to over 396.5 per 100,000 population in men and 236.3 per 100,000 population in Section 1 5 women aged 75 years and above, ONS (2005). The median age of patients at diagnosis is 72 NBOCAP (2005). There is not only age- and sex-specific incidence, but also a regional specific incidence, which shows a range per 100,000 from 41.4 in London to 77.6 in the North East for men and from 36.4 in London to 61.1 in the South West for women. These differences reflect not just lifestyle and environmental factors, but also the underlying demography, with London having a younger population.

SURVIVAL Prognosis is strongly related to the stage of cancer at diagnosis; late-stage cancers are associated with poorer survival, more intensive and disfiguring treatments, and increased morbidity. Furthermore, patients diagnosed at an earlier stage are more likely to undergo successful resection and may be cured. Importantly, the treatment of patients with bowel cancer focuses not just on improving survival, but also on morbidity. Whilst surgery and subsequent adjuvant therapies are often associated with favourable outcomes, many patients will eventually develop advanced disease and distant metastases, which typically present within two years of the initial treatment. In around 15% of cases, patients will present with advanced disease, and 50% of these patients will present with liver metastases. Based on published sources, the overall five-year survival rate for patients with colon cancer is reported to be 47.6% for men and 47.4% for women. The overall five-year survival rate for patients with rectal cancer is estimated to be 48.7% for men and 51.35% for women based on data for people diagnosed between 1996 and 1999. Coleman 2004.

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XXIX - ECONOMIC AND FINANCIAL ANALYSIS OF ENGLISH PRESS FOR THE CRC A. Ferreira-Alemao, MD PhD UNIVERSIDAD COMPLUTENSE DE MADRID

Cost of bowel cancer £1.6 billion A recent study by Oxford University researchers has revealed that the heath and economic cost of bowel cancer in the UK is more than prostate cancer and breast cancer. In terms of economic losses, healthcare costs and the burden of unpaid care provided by friends and family, cancer costs the UK £15 billion a year.

Bowel cancer totals £1.6bn compared to £2.4bn for lung cancer, £1.5bn for breast cancer and £800m for prostate cancer. More than half of this figure relates to people being unable to work and careers taking time off work to care for their friends and family.

Bowel Cancer Screening cuts deaths A bowel cancer screening programme in England is on course to cut deaths by a sixth, say researchers studying results from the first million people tested. However, the work, published in the journal Gut, has raised concerns that the programme, launched in 2006, misses tumours in certain parts of the colon. Testers checked a faeces sample for signs of abnormal bleeding. The researcher who analysed the results said money should be spent on bringing in more sensitive tests. The screening programme aims to catch the tumours earlier, meaning more patients can be cured. Several million people aged 69 and over have now been screened, with approximately half of those invited taking part. When results from the first 1.08 million taking part were collated, it was found that 2.5% of men and 1.5% of women had received an abnormal result, and were sent for further tests, usually colonoscopy, in which a tube with a camera on the end is passed into the colon. Among the men with abnormal results, 43% turned out to have either cancer or pre-cancerous growths in their colon.

The figure for women was 11.6%.Most of these were early-stage cancers, which are generally easier to treat.

Professor Julietta Patnick, director of the NHS Cancer Screening Programmes, said she was "delighted" by the results. "We are pleased to be on track to cut bowel cancer deaths by our target of 16% - early detection is crucial to lowering the number deaths from bowel cancer." However, the test appeared to be less effective at spotting cancers in the upper section, or "right side" of the colon. Cancer statistics from millions of people diagnosed with the disease suggest that for every three cancers in the left side of the colon, there will be one in the right side. However, of the thousands of cancers found by the screening test, only 14% were on the right side. Prof Richard Logan, of Nottingham University, who led the study, said it was not yet fully understood why cancers on the right-hand side were not showing up during screening.

Among the possibilities, he said, were that they grew faster, and were more likely to be diagnosed conventionally, or that they were less likely to bleed and trigger a positive test result. However, he said that the current blood test was "old fashioned", and should be replaced by a more modern alternative which might be 236

RIBOGRAMA PROJECT more sensitive. He said: "There is a much better test available, but it will cost money to introduce it, and that money is not currently there." A spokesman for the screening programme said that the move to the new test was being "actively considered", although no date had been set for a change. Deborah Alsina, the chief executive of Bowel Cancer UK, welcomed the results, adding that separate moves to widen access to a form of colonoscopy called flexible sigmoidoscopy would also improve screening. "As a higher proportion of cancers are being found in the left side of the bowel than predicted, the rollout of flexible sigmoidoscopy screening in the new year will help to save even more lives. "However, this does need to be rolled out quickly with a sustained focus on areas with low uptake of screening."

People over 60 urged to have regular screening Bowel Cancer UK's statement in relation to the West Midlands Cancer Intelligence Unit press release that people aged over 60 have been urged to have regular bowel cancer screenings after scientists found those who did had a better chance of survival. Deborah Alsina, CEO, Bowel Cancer UK said: "This research highlights the importance of screening. It does work and it's the most effective tool for detecting cancer early especially among those most at risk i.e. people over 60. The problem is that only around 50% of people return their kits, and more worrying still is that in some areas that number is significantly lower. It is vitally important that we identify a better way of encouraging people to take part in screening. I can't stress enough how important it is for people to take part, it might save your life."

West Midlands Cancer Intelligence Unit: People aged over 60 have been urged to have regular bowel cancer screenings after scientists found those who did had a better chance of survival. Experts said those who performed the test at home and went to subsequent appointments were more likely to be diagnosed at an earlier stage than those diagnosed from their symptoms.

Bowel cancer is the third most common cancer in both men and women, with around 40,000 people diagnosed with the disease each year. Researchers looked at people aged 60 to 69 who were diagnosed with the disease in the West Midlands between January 2006 and September 2011. They compared the stage at diagnosis in patients picked up at screening compared to those diagnosed from symptoms.

They found that 18.5 per cent of bowel cancers detected through screening were at the earliest stages compared with 9.4 per cent of cancers diagnosed through symptomatic routes.

Sam Johnson, lead researcher based at the West Midlands Cancer Intelligence Unit, said: 'When bowel cancer is diagnosed at an earlier stage, it's easier to treat, has a lower chance of coming back and better survival rates. “Our research shows that screening can play an important role in improving bowel cancer survival by picking up cancers at an earlier stage. The findings were presented at the National Cancer Intelligence Network (NCIN) conference in Birmingham”.

NCIN head, Chris Carrigan said: “When bowel cancer is found at the earliest stage, there is an excellent chance of survival, with more than 90 per cent of people surviving the disease at least five years. 'This study highlights the potential improvements we can make through encouraging more people to take up their screening invitation so the disease is diagnosed earlier.”

Cancer Research UK head of health information and evidence, Hazel Nunn added: “Bowel screening uptake is worryingly low, particularly amongst men. And this is a useful reminder for older people to complete their bowel screening kit when it arrives in the post.”

The NHS Bowel Cancer Screening Programme offers screening every two years to all men and women aged 60 to 69. People in this age group are sent an invitation followed by a screening kit, so they can do the test at 237

RIBOGRAMA PROJECT home. It involves wiping small stool samples onto the testing card, which is then sealed and sent in the post to a lab for testing. The test detects tiny amounts of blood, which you cannot normally see, as polyps and bowel cancers sometimes bleed. So the test does not diagnose the disease but will tell you if you need a further examination by a doctor. Around 98 per cent of samples are judged as 'normal' although this does not guarantee that a person does not have bowel cancer.

Bowel cancer patients diagnosed through screening more likely to survive Bowel cancer patients whose disease was found through screening have a better chance of beating their disease than those diagnosed after developing symptoms, new research shows today (Wednesday).

The study, published in the British Journal of Cancer, also adds to evidence that the test used in bowel screening - which looks for blood in stool samples - is better at finding bowel cancers in men, and in the lower part of the bowel.

While the blood test - known as FOBt - has been shown to be effective, it is not flawless. The study found that in people who attended screening nearly a quarter of cancers were diagnosed in between tests - suggesting these tumours were either missed by FOBt or these cancers were particularly fast-growing and developed in the two years between screening tests. The study findings, based on north-east England, support Cancer Research UK's calls to ensure the bowel screening programmes are as effective as possible. This could be done by including a better stool blood test and implementing the Flexi-Scope bowel screening test swiftly.

Using data from the Northern Colorectal Cancer Audit Group in north-east England, researchers looked at more than 1,300 bowel cancers diagnosed between April 2007 and March 2010. The results show that nearly 40 per cent of all screen-detected cancers are at an early stage with an improved survival rate compared to cancers found in patients who did not attend screening. Cancer Research UK figures show that when bowel cancer is found at the earliest stage, more than 90 per cent of people survive their disease at least five years.

Dr Michael Gill, lead author of the study based at the Wansbeck General Hospital in Northumberland and Durham University, said: "Compared to the trials which led to the introduction of the national bowel screening programme, our research shows that the proportion of bowel cancers detected through screening has improved with the roll-out of national screening. But too many bowel cancers are slipping through the net. We need to understand why the present blood test is failing to pick up cancers in certain parts of the bowel, and in women."

The Scottish and English bowel screening programmes are considering a more effective blood test called Faecal Immunochemical Test (FIT) - which is more efficient at detecting hidden traces of blood in stool samples. And in 2012, the screening programme in England will also begin to include the new Flexi-Scope test. Sarah Woolnough, Cancer Research UK's director of policy, said: "There is persuasive evidence that the new blood test, FIT, is a more effective test for bowel screening. The test also requires patients to provide fewer stool samples and so is less complicated to complete and return - which we hope will improve take-up of bowel screening.

"Cancer Research UK is pleased that England will add the Flexi-Scope test to its bowel screening programme but the roll-out needs to be rapid. We need ongoing monitoring and resource to ensure the roll-out runs to time and plan. While we understand that Scotland, Wales and Northern Ireland will learn from the English pilots, we urge them to begin planning their own roll-out of the test to avoid undue delays and ultimately save more lives.

Compared with breast and cervical screening, bowel screening uptake is worryingly low, particularly among men. This study is an important reminder for people to complete their bowel screening kit when it arrives in the post."

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RIBOGRAMA PROJECT Bowel cancer is the third most common cancer in the UK with more than 41,000 people diagnosed with the disease each year - over 100 people each day. The bowel screening programme has only been fully up and running in England since 2010 but it is thought it will eventually save around 2,000 lives each year in the UK.

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XXX - MEDICATIONS WITH ANTIPROLIFERATIVE PROPRIETIES The available anticancer drugs have distinct mechanisms of action which may vary in their effects on different types of normal and cancer cells. A single "cure" for cancer has proved elusive since there is not a single type of cancer but as many as 100 different types of cancer. In addition, there are very few demonstrable biochemical differences between cancerous cells and normal cells. For this reason the effectiveness of many anticancer drugs is limited by their toxicity to normal rapidly growing cells in the intestinal and bone marrow areas. A final problem is that cancerous cells which are initially suppressed by a specific drug may develop a resistance to that drug. For this reason cancer chemotherapy may consist of using several drugs in combination for varying lengths of time.

Cancer Chemotherapy: Chemotherapy drugs, are sometimes feared because of a patient's concern about toxic effects. Their role is to slow and hopefully halt the growth and spread of a cancer. There are three goals associated with the use of the most commonly-used anticancer agents.

1. Damage the DNA of the affected cancer cells.

2. Inhibit the synthesis of new DNA strands to stop the cell from replicating, because the replication of the cell is what allows the tumor to grow.

3. Stop mitosis or the actual splitting of the original cell into two new cells. Stopping mitosis stops cell division (replication) of the cancer and may ultimately halt the progression of the cancer.

Unfortunately, the majority of drugs currently on the market are not specific, which leads to the many common side effects associated with cancer chemotherapy. Because the common approach of all chemotherapy is to decrease the growth rate (cell division) of the cancer cells, the side effects are seen in bodily systems that naturally have a rapid turnover of cells iincluding skin, hair, gastrointestinal, and bone marrow. These healthy, normal cells, also end up damaged by the chemotherapy program.

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XXXI - APTAMERS Structure of an RNA aptamer specific for biotin.

The aptamer surface and backbone are shown in yellow. Biotin (spheres) fits snugly into a cavity of the RNA surface

APTAMERS (from the Latin aptus - fit, and Greek meros - part) are oligonucleic acid or peptide molecules that bind to a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. Aptamers can be combined with ribozymes to self-cleave in the presence of their target molecule. These compound molecules have additional research, industrial and clinical applications.

More specifically, aptamers can be classified as:  DNA or RNA or XNA aptamers. They consist of (usually short) strands of oligonucleotides.  Peptide aptamers. They consist of a short variable peptide domain, attached at both ends to a protein scaffold.

NUCLEIC ACID APTAMERS Nucleic acid aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.

Aptamers are useful in biotechnological and therapeutic applications as they offer molecular recognition properties that rival that of the commonly used biomolecule, antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.

In 1990, two labs independently developed the technique of selection: the Gold lab, using the term SELEX for their process of selecting RNA ligands against T4 DNA polymerase; and the Szostak lab, coining the term in vitro selection, selecting RNA ligands against various organic dyes. The Szostak lab also coined the term aptamer (from the Latin, apto, meaning ‘to fit’) for these nucleic acid-based ligands. Two years later, the Szostak lab and Gilead Sciences, independent of one another, used in vitro selection schemes to evolve single stranded DNA ligands for organic dyes and human coagulant, thrombin, respectively. There does not appear to be any systematic differences between RNA and DNA aptamers, save the greater intrinsic chemical stability of DNA. Interestingly enough, the notion of selection in vitro was actually preceded twenty-plus years prior when Sol Spiegelman used a Qbeta replication system as a way to evolve a self-replicating molecule.[1] In addition, a year before the publishing of in vitro selection and SELEX, Gerald Joyce used a system that he termed ‘directed evolution’ to alter the cleavage activity of a ribozyme. Since the discovery of aptamers, many researchers have used aptamer selection as a means for application and discovery. In 2001, the process of in vitro selection was automated by the Ellington lab at the University of Texas at Austin, and at SomaLogic, Inc (Boulder, CO), reducing the duration of a selection experiment from six weeks to three days. While the process of artificial engineering of nucleic acid ligands is highly interesting to biology and biotechnology, the 241

RIBOGRAMA PROJECT notion of aptamers in the natural world had yet to be uncovered until 2002 when two groups led by Ronald Breaker and Evgeny Nudler discovered a nucleic acid-based genetic regulatory element (which was named riboswitch) that possesses similar molecular recognition properties to the artificially made aptamers. In addition to the discovery of a new mode of genetic regulation, this adds further credence to the notion of an ‘RNA World,’ a postulated stage in time in the origins of life on Earth.

Both DNA and RNA aptamers show robust binding affinities for various targets.[2][3][4] DNA and RNA aptamers have been selected for the same target. These targets include lysozyme,[5] thrombin,[6] human immunodeficiency virus trans-acting responsive element (HIV TAR),[7] hemin,[8] interferon γ,[9] vascular endothelial growth factor (VEGF),[10] prostate specific antigen (PSA),[11] [12]and dopamine.[13] In the case of lysozyme, HIV TAR, VEGF and dopamine the DNA aptamer is the analog of the RNA aptamer, with thymine replacing uracil. The hemin, thrombin, and interferon γ, DNA and RNA aptamers were selected through independent selections and have unique sequences. Considering that not all DNA analogs of RNA aptamers show functionality the correlation between DNA and RNA sequence and their structure and function requires further investigation. Lately, a concept of smart aptamers, and smart ligands in general, has been introduced.

It describes aptamers that are selected with pre-defined equilibrium (), rate (, ) constants and thermodynamic (ΔH, ΔS) parameters of aptamer-target interaction. Kinetic capillary electrophoresis is the technology used for the selection of smart aptamers. It obtains aptamers in a few rounds of selection.

Recent developments in aptamer-based therapeutics have been rewarded in the form of the first aptamerbased drug approved by the U.S. Food and Drug Administration (FDA) in treatment for age-related macular degeneration (AMD), called Macugen offered by OSI Pharmaceuticals. In addition, the company NeoVentures Biotechnology Inc. (http://www.neoventures.ca) has successfully commercialized the first aptamer based diagnostic platform for analysis of mycotoxins in grain. Many contract companies develop aptamers and aptabodies to replace antibodies in research, diagnostic platforms, drug discovery, and therapeutics.

Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, mainly due to nuclease degradation and clearance from the body by the kidneys, a result of the aptamer's inherently low molecular weight. Unmodified aptamer applications currently focus on treating transient conditions such as blood clotting, or treating organs such as the eye where local delivery is possible. This rapid clearance can be an advantage in applications such as in vivo diagnostic imaging. An example is a tenascin-binding aptamer under development by Schering AG for cancer imaging. Several modifications, such as 2'-fluorine-substituted pyrimidines, polyethylene glycol (PEG) linkage, etc. (both of which are used in Macugen, an FDA-approved aptamer) are available to scientists with which to increase the serum half-life of aptamers easily to the day or even week time scale. Another approach to increase the nuclease resistance of aptamers is to develop Spiegelmers, which are composed entirely of an unnatural L-ribonucleic acid backbone. A spiegelmer of the same sequence has the same binding properties of the corresponding RNA aptamer, except it binds to the mirror image of its target molecule.

In addition to the development of aptamer-based therapeutics, many researchers such as the Ellington lab and independently another company SomaLogic (Boulder, CO) have been developing diagnostic techniques for aptamer based plasma protein profiling called aptamer plasma proteomics. This technology will enable future multi-biomarker protein measurements that can aid diagnostic distinction of disease versus healthy states.

Somalogic aptamers to HER2 and EGFR membrane proteins have also been demonstrated to work in the context of rapid frozen tissue intraoperative diagnostic pathology applications. [14] As a resource for all in vitro selection and SELEX experiments, the Ellington lab has developed the Aptamer Database cataloging all published experiments. This is found at http://aptamer.icmb.utexas.edu/.

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RIBOGRAMA PROJECT Peptide aptamers Peptide aptamers are proteins that are designed to interfere with other protein interactions inside cells. They consist of a variable peptide loop attached at both ends to a protamersein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody's (nanomolar range). The variable loop length is typically composed of ten to twenty amino acids, and the scaffold may be any protein which has good solubility and compacity properties. Currently, the bacterial protein Thioredoxin-A is the most used scaffold protein, the variable loop being inserted within the reducing active site, which is a -Cys-Gly-Pro-Cys- loop in the wild protein, the two Cysteines lateral chains being able to form a disulfide bridge. Peptide aptamer selection can be made using different systems, but the most used is currently the yeast two-hybrid system. Selection of Ligand Regulated Peptide Aptamers (LiRPAs) has been demonstrated. By displaying 7 amino acid peptides from a novel scaffold protein based on the trimeric FKBPrapamycin-FRB structure, interaction between the randomized peptide and target molecule can be controlled by the small molecule Rapamycin or non-immunosuppressive analogs.

Peptide aptamer can also be selected from combinatorial peptide libraries constructed by phage display and other surface display technologies such as mRNA display, ribosome display, bacterial display and yeast display. These experimental procedures are also known as biopannings. Among peptides obtained from biopannings, mimotopes can be considered as a kind of peptide aptamers. All the peptides panned from combinatorial peptide libraries have been stored in a special database with the name MimoDB,[15] which is freely available at http://immunet.cn/mimodb.

AptaBiD AptaBiD or Aptamer-Facilitated Biomarker Discovery is a technology for biomarker discovery.[16] AptaBiD is based on multi-round generation of an aptamer or a pool of aptamers for differential molecular targets on the cells which facilitates exponential detection of biomarkers. It involves three major stages: (i) differential multiround selection of aptamers for biomarker of target cells; (ii) aptamer-based isolation of biomarkers from target cells; and (iii) mass spectrometry identification of biomarkers. The important feature of the AptaBiD technology is that it produces synthetic affinity probes (aptamers) simultaneously with biomarker discovery. In AptaBiD, aptamers are developed for cell surface biomarkers in their native state and conformation. In addition to facilitating biomarker identification, such aptamers can be directly used for cell isolation, cell visualization, and tracking cells in vivo. They can also be used to modulate activities of cell receptors and deliver different agents (e.g., siRNA and drugs) into the cells.

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References 1. Mills, DR; Peterson, RL; Spiegelman, S (1967 Jul). "An extracellular Darwinian experiment with a selfduplicating nucleic acid molecule.". Proceedings of the National Academy of Sciences of the United States of America 58 (1): 217–24. PMID 5231602.

2. Neves, M.A.D.; O. Reinstein, M.Saad, P.E. Johnson (2010). "Defining the secondary structural requirements of a cocaine-binding aptamer by a thermodynamic and mutation study". Biophys Chem 153: 9–16. doi:10.1016/j.bpc.2010.09.009. PMID 21035241.

3. Baugh, C.; D. Grate, C.Wilson (2000). "2.8 angstrom crystal structure of the malachite green aptamer.". J. Mol. Biol. 301: 117–128. doi:10.1006/jmbi.2000.3951. PMID 10926496.

4. Dieckmann, T.; E. Fujikawa, X. Xhao, J. Szostak, J. Feigon (1995). "Structural Investigations of RNA and DNA aptamers in Solution". Journal of Cellular Biochemistry: 56–56.

5. Potty, A.; K. Kourentzi, H. Fang, G. Jackson, X. Zhang, G. Legge, R. Willson (2009). "Biophysical Characterization of DNA Aptamer Interactions with Vascular Endothelial Growth Factor.". Biopolymers 91: 145–156. doi:10.1002/bip.21097. PMID 19025993.

6. Long, S.; M. Long, R. White, B. Sullenger (2008). "Crystal structure of an RNA aptamer bound to thrombin". RNA 14 (2): 2504–2512. PMID 18971322.

7. F.; S. Reigadas, J. Hansen, H. Orum, C. Di Primo, J. Toulme (2006). "Aptamers targeted to an RNA hairpin show improved specificity compared to that of complementary oligonucleotides.". Biochemistry 45: 12076–12082. doi:10.1021/bi0606344. PMID 17002307.

8. Liu, M.; T. Kagahara, H. Abe, Y. Ito (2009). "Direct In Vitro Selection of Hemin-Binding DNA Aptamer with Peroxidase Activity". Bulletin of the Chemical Society of Japan 82: 99–104.

9. Min, K.; M. Cho, S. Han, Y. Shim, J. Ku, C. Ban (2008). "A simple and direct electrochemical detection of interferon-gamma using its RNA and DNA aptamers.". Biosensors & Bioelectronics 23: 1819–1824. doi:10.1016/j.bios.2008.02.021. PMID 18406597.

10. Ng, E.W.M; D.T. Shima, P. Calias, E.T. Cunningham, D.R. Guyer, A.P. Adamis (2006). "Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease.". Nature Reviews Drug Discovery 5 (2): 123– 132. doi:10.1038/nrd1955. PMID 16518379.

11. Savory, N.; K. Abe, K. Sode, K. Ikebukuro (2010). "Selection of DNA aptamer against prostate specific antigen using a genetic algorithm and application to sensing.". Biosensors & Bioelectronics 15: 1386– 91. doi:10.1016/j.bios.2010.07.057. PMID 20692149.

12. Jeong, S.; S.R. Han, Y.J. Lee, S.W. Lee (2010). "Selection of RNA aptamers specific to active prostate-specific antigen.". Biotechnology Letters 32: 379–85. doi:10.1007/s10529-009-0168-1. PMID 19943183.

13. Walsh, R.; M. DeRosa (2009). "Retention of function in the DNA homolog of the RNA dopamine aptamer.". Biochemical and Biophysical Research Communications 388: 732–735. doi:10.1016/j.bbrc.2009.08.084. PMID 19699181.

14. Gupta S, Thirstrup D, Jarvis TC, Schneider DJ, Wilcox SK, Carter J, Zhang C, Gelinas A, Weiss A, Janjic N, Baird GS. (Jan 2011). "Rapid Histochemistry Using Slow Off-rate Modified Aptamers With Anionic Competition.". Applied Immunohistochemistry & Molecular Morphology 19 (1): Epub ahead of print. doi:10.1097/PAI.0b013e3182008c29. PMID 21217521.

15. Huang, J; Ru, B, Zhu, P, Nie, F, Yang, J, Wang, X, Dai, P, Lin, H, Guo, FB, Rao, N (2011-11-03).

"MimoDB 2.0: a mimotope database and beyond.". Nucleic Acids Research 40 (1): D271–7. doi:10.1093/nar/gkr922. PMC 3245166. PMID 22053087.

16. Berezovski MV, Lechmann M, Musheev MU, Mak TW, Krylov SN (Jul 2008). "Aptamer-facilitated biomarker discovery (AptaBiD)". J Am Chem Soc. 130 (28): 9137–43. doi:10.1021/ja801951p. PMID 18558676.

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Further reading  

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Ellington AD, Szostak JW (Aug 1990). "In vitro selection of RNA molecules that bind specific ligands". Nature 346 (6287): 818–22. doi:10.1038/346818a0. PMID 1697402.

Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ (Feb 1992). "Selection of single-stranded DNA molecules that bind and inhibit human thrombin". Nature 355 (6360): 564–6. doi:10.1038/355564a0. PMID 1741036.

Hoppe-Seyler F, Butz K (2000). "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 426–30. doi:10.1007/s001090000140. PMID 11097111.

Carothers JM, Oestreich SC, Davis JH, Szostak JW (Apr 2004). "Informational complexity and functional activity of RNA structures". J Am Chem Soc. 126 (16): 5130–7. doi:10.1021/ja031504a. PMID 15099096.

Cohen BA, Colas P, Brent R (Nov 1998). "An artificial cell-cycle inhibitor isolated from a combinatorial library".

Proc Natl Acad Sci USA. 95 (24): 14272–7. doi:10.1073/pnas.95.24.14272. PMC 24363. PMID 9826690.

Binkowski BF, Miller RA, Belshaw PJ (Jul 2005). "Ligand-regulated peptides: a general approach for modulating protein-peptide interactions with small molecules".

Chem Biol.

12 (7): 847–55. doi:10.1016/j.chembiol.2005.05.021. PMID 16039531.

Sullenger BA, Gilboa E (Jul 2002). "Emerging clinical applications of RNA". Nature 418 (6894): 252–8. doi:10.1038/418252a. PMID 12110902.

Ng EW, Shima DT, Calias P, Cunningham ET, Guyer DR, Adamis AP (Feb 2006). "Pegaptanib, a targeted antiVEGF aptamer for ocular vascular disease". Nat Rev Drug Discov 5 (2): 123–32. doi:10.1038/nrd1955. PMID 16518379.

Drabovich AP, Berezovski M, Okhonin V, Krylov SN (May 2006). "Selection of smart aptamers by methods of kinetic capillary electrophoresis". Anal Chem. 78 (9): 3171–8. doi:10.1021/ac060144h. PMID 16643010.

Cho EJ, Lee JW, Ellington, AD (2009). "Applications of Aptamers as Sensors". Annual Review of Analytical Chemistry 2 (1): 241–64. doi:10.1146/annurev.anchem.1.031207.112851. PMID 20636061.

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XXXII - CANCER-SELECTIVE ANTIPROLIFERATIVE ACTIVITY IS A GENERAL PROPERTY OF SOME GRICH OLIGODEOXYNUCLEOTIDES Enid W. Choi1, Lalitha V. Nayak2 and Paula J. Bates*,1,2 + Author Affiliations 1Department of Biochemistry and Molecular Biology and 2Department of Medicine, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA *To whom correspondence should be addressed. Tel: +1 502 852 2432; Fax: +1 502 852 2356; Email: paula.bates@louisville.edu Received June 9, 2009.

Revision received October 23, 2009.

Accepted November 8, 2009.

ABSTRACT Oligodeoxynucleotide libraries containing randomly incorporated bases are used to generate DNA aptamers by systematic evolution of ligands by exponential enrichment (SELEX). We predicted that combinatorial libraries with alternative base compositions might have innate properties different from the standard library containing equimolar A + C + G + T bases. In particular, we hypothesized that G-rich libraries would contain a higher proportion of quadruplex-forming sequences, which may impart desirable qualities, such as increased nuclease resistance and enhanced cellular uptake. Here, we report on 11 synthetic oligodeoxynucleotide libraries of various base combinations and lengths, with regard to their circular dichroism, stability in serumcontaining medium, cellular uptake, protein binding and antiproliferative activity. Unexpectedly, we found that some G-rich libraries (composed of G + T or G + C nucleotides) strongly inhibited cancer cell growth while sparing non-malignant cells. These libraries had spectral features consistent with G-quadruplex formation, were significantly more stable in serum than inactive libraries and showed enhanced cellular uptake. Active libraries generally had strong protein binding, while the pattern of protein binding suggested that G/T and G/C libraries have distinct mechanisms of action. In conclusion, cancer-selective antiproliferative activity may be a general feature of certain G-rich oligodeoxynucleotides and is associated with quadruplex formation, nuclease resistance, efficient cellular uptake and protein binding.

INTRODUCTION Cancer is a leading cause of death throughout the world. Although many drugs have been developed to treat cancer, the majority of the oncology pharmacopeia causes serious, dose-limiting side effects due to damage to 246

RIBOGRAMA PROJECT healthy cells. There exists a clear need for better, more specific cancer therapies, not only to increase efficacy against cancer cells but also to decrease the suffering of patients who are subjected to these drug regimens.

Nucleic acid aptamers are emerging as a new class of targeted therapeutic agents and are the focus of a rapidly growing field of medical research (1–4). These small synthetic oligomers of DNA or RNA form stable three-dimensional structures and bind to defined molecular targets via shape-specific recognition. Thus, in essence, their mechanism of action is similar to that of protein-based monoclonal antibodies. However, due to their smaller size and different chemical composition, aptamers have a number of potential advantages over antibodies. These include ease of manufacture and storage, facile conjugation, better tumor penetration, more rapid systemic clearance and non-immunogenicity. In recent years, aptamers have been identified as potential treatments for a variety of diseases, including macular degeneration (5,6), autoimmune disease (7), disseminated intravascular coagulation (8,9) and prion diseases (10). In the oncology field, the most clinically advanced aptamer, and the first anticancer aptamer to be tested in humans, is a 26-nt G-rich DNA known as AS1411 (formerly, AGRO100). This nucleolin-targeted aptamer, which was discovered by our group, has shown promising clinical activity without any major side effects in Phase I and ongoing Phase II clinical trials (11).

New aptamers against specific targets are most often engineered via systematic evolution of ligands by exponential enrichment (SELEX) (1,12,13), an iterative methodology designed to create target-specific aptamers from a combinatorial library (Figure 1A). For DNA aptamers, a single-stranded oligodeoxynucleotide library consisting of two known primer-binding sequences flanking a random sequence of usually 20–40 nt in length is applied to the target of interest. The molecules that do not bind are washed away, and the remainder are harvested and amplified by polymerase chain reaction (PCR). The aptamer-enriched pool of singlestranded molecules is then isolated from the PCR product, reapplied to the target and the process is repeated.

In this manner, highly specific aptamer sequences are usually evolved after 10–20 cycles. For RNA aptamers, a similar procedure with additional steps for transcription and reverse transcription can be utilized. Typically, the target of interest for SELEX is a purified protein or small molecule, but the development of aptamers against intact cells or organisms can be also achieved (1,14).

OVERVIEW OF SELEX PROCEDURE AND OLIGONUCLEOTIDE LIBRARIES (A) Schematic of SELEX procedure. (B) Diagram of N library. Primer-binding regions (PBR) flank a length of random sequence, for which each base has equal likelihood of being an A, C, G or T. In theory, the library contains 4n different sequences, where n is the length of the random sequence (in this example n = 26). (C) Example of a guanine-rich GT library. The random sequence is restricted such that each base may be either a G or a T. This library should contain 2n different sequences (in this example n = 26).

One shortcoming of traditional SELEX is the high number of iterations required, which consumes time and resources, and increases the likelihood of artifacts (since every round of SELEX requires PCR and must be carefully protected from contamination). Moreover, for in vivo use, evolved sequences must typically be modified to enhance their pharmacokinetic properties and to increase resistance to serum nucleases. This can be achieved post-SELEX, or by incorporating modified bases or backbones during the SELEX procedure (1,15). However, these approaches have some drawbacks: post-SELEX modification can reduce aptamer activity or increase toxicity, and not all modified nucleotides are compatible with the polymerases used during SELEX. An additional shortcoming of aptamers (which is also shared by antibodies) is their inefficient cellular internalization, which generally limits their clinical targets to extracellular or cell surface molecules.

In an attempt to address some of these limitations, we have considered the use of alternative starting libraries for SELEX. Rather than the random N library containing billions of sequences, the vast majority of which will 247

RIBOGRAMA PROJECT not turn out to be clinically useful aptamers, we propose beginning with smaller libraries, especially those which may be enriched in molecules that contain secondary structure motifs, such as hairpins or G-quartets. In particular, we hypothesized that libraries enriched in guanine (G) residues would contain a higher proportion of quadruplex-forming sequences, which may impart increased functionality, nuclease resistance and cellular uptake compared to the N library. An additional driving force for this study was the realization that very G-rich sequences such as AS1411 (5′-d[GGTGGTGGTGGTTGTGGTGGTGGTGG]), which was discovered by chance, would rarely occur in the standard N library, making it unlikely to have been discovered by standard SELEX. Furthermore, our previous work involving the ‘bench-to-bedside’ development of AS1411 and the preclinical research of several other groups who are researching a variety of different G/T-containing oligonucleotides (11) led us to focus on G/T DNA libraries for our studies on the effect of oligonucleotide length.

As a prelude to SELEX studies using alternative oligodeoxynucleotide libraries, we have sought to establish the baseline characteristics of various libraries and to test our hypothesis that G-rich libraries will be enriched in quadruplex-forming sequences with improved nuclease resistance and cellular uptake. In this project, we have examined 11 oligodeoxynucleotide libraries of varying base compositions and lengths, as well as three monobasic oligomers. We have performed studies to evaluate various characteristics of the libraries, including a well-established colorimetric assay to determine their intrinsic antiproliferative activities in three different cancer cell lines compared to non-malignant cells. As far as we know, we are the first to perform such a comprehensive screen of oligonucleotide libraries. In addition, we report here the unexpected discovery that certain G-rich libraries have high levels of intrinsic, cancer-selective antiproliferative activity.

MATERIALS AND METHODS OLIGODEOXYNUCLEOTIDES AND LIBRARIES All oligonucleotides and oligonucleotide libraries were purchased from Integrated DNA Technologies, Inc. (Coralville, IA, USA) and are shown in Figure 2. The relative proportion of bases was specified for libraries, which can be synthesized by mixing phosphoramidite precursors in the desired ratio. The specified bases are then randomly incorporated into the sequence. Lyophilized libraries were resuspended in filter-sterilized water, then heated at 65°C for 10 min and vortexed vigorously to dissolve. For uptake studies, three thymidine bases (TTT) were added to the 3′-end of the library sequence to avoid quenching the 3′-fluorophore, which can occur when it is proximal to a G-quartet (Choi,E., Nayak,L. and Bates,P. unpublished data). A G26 oligodeoxynucleotide was originally proposed for study, but is not commercially available due to difficulties in synthesis of oligodeoxyguanosines of this length.

CELL CULTURE Four cell lines were comprehensively studied: A549 lung adenocarcinoma, DU145 prostate adenocarcinoma, MCF7 mammary carcinoma and Hs27 non-malignant skin fibroblasts. All cell lines were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in complete cell culture medium of Dulbecco's; Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-treated (15 min at 65°C) fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Invitrogen). The MCF7 cell line alone was grown using charcoal-stripped serum (Invitrogen). All cells were cultured at 37°C in 5% CO2. Methods for cell culture and antiproliferative assays using MCF10A cells can be found in the Supplementary Data.

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RIBOGRAMA PROJECT ANTIPROLIFERATIVE ACTIVITY ASSAY In the four cells lines, each library/oligomer was tested in triplicate at eight concentrations of oligonucleotide (0, 2, 4, 6, 8, 10, 12 and 14 μM) using a modified previously published MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] assay protocol (16). Briefly, cells were seeded in 96-well tissue culture plates and incubated overnight. To account for intrinsic differences in growth rate, the A549 and DU145 lines were plated at 1000 cells per well, MCF7 at 2000 per well and Hs27 at 3000 per well. Oligomer library stock solutions were then added to each well to yield the desired final concentration. Plates were incubated with the oligomer libraries for 5 days, during which the cell culture medium was not changed. On Day 5, cell viability was determined and the background corresponding to medium alone (no cells) was subtracted. For each concentration, the mean and standard error of triplicate wells was plotted. Assays were repeated at least twice in triplicate to ensure reproducibility of the results.

CIRCULAR DICHROISM SPECTROSCOPY Oligomer library stocks were diluted to a concentration of 5 µM (strand concentration) using either water (buffer was not included in order to be representative of the stock solutions used to treat cells), or aqueous solutions containing 100 mM sodium chloride or 100 mM potassium chloride. Samples were incubated at 95°C for 5 min, then cooled on ice until analysis. Circular dichroism (CD) spectra were collected at 20°C using a Jasco J-715 spectropolarimeter between 340 and 220 nm wavelengths, using 200 nm/min scanning speed, 2 s response time, 5 nm bandwidth and 1-cm pathlength. For each set of samples, the same solution used to prepare the oligomers was used as the reference. The resulting spectra in millidegrees (mdeg) were converted to molar CD, referring to per mole of nucleotide. Methods for analysis of (dC)26 at various pH can be found in the Supplementary Data.

NUCLEASE-RESISTANCE IN SERUM-CONTAINING MEDIUM Each oligomer library was 5′-end-labeled with 32P using T4 kinase (Invitrogen) and unincorporated γ-32P-ATP was removed from samples using TE-Midi SELECT-D G-25 columns (Shelton Scientific, Shelton, CT, USA), according to manufacturers’ directions. Collection tubes containing end-labeled libraries were kept on ice, or stored at −20°C. The radioactivity of end-labeled libraries was measured in a scintillation counter on the day of the experiment. For each library to be tested, four reaction tubes containing complete cell culture medium without antibiotics were prepared. One million counts per minute of end-labeled oligomer was added to each of four tubes. Samples were incubated at 37°C, and tubes were flash-frozen in a dry ice/ethanol bath at 0, 1.5, 24 and 72 h. Frozen samples were stored at −80°C until all samples had been harvested. Samples were then thawed on ice and run on 12% denaturing polyacrylamide gels. Band density was measured over each lane using the UN-SCAN-IT gel densitometry application (Silk Scientific, Orem, UT, USA). The percent sample remaining was calculated taking the signal of the zero time point lane to be 100%.

CELLULAR UPTAKE Uptake experiments were performed using oligomer libraries labeled at the 3′ end with FAM (6carboxyfluorescein) for representative libraries that were inactive (N 26mer and GA 26mer) or active (GC 26mer and GT 26mer). For each sample, 3 × 105 cells were plated and incubated overnight. The following day, fresh medium was added to each plate. FAM-labeled libraries were added to make a final concentration of 5-µM oligonucleotide and plates were incubated for 1 h. The medium containing unbound oligo was removed. Cells were washed twice and treated with trypsin (Invitrogen) to remove surface-bound 249

RIBOGRAMA PROJECT oligonucleotides and harvest cells. Cells were kept on ice and protected from light until analysis. Propidium iodide (PI, 2 µg per sample, BD Biosciences, San Jose, CA, USA) was added as a marker of viability. Ten thousand cells were counted, after gating to exclude PI-positive (non-viable) cells from analysis. Cells were analyzed using a FACSCalibur cytometer (BD Biosciences). The relative fluorescence of cells was determined using the FlowJo software program (Tree Star, Ashland, OR, USA).

PROTEIN BINDING USING ELECTROPHORETIC MOBILITY SHIFT ASSAY Libraries were end-labeled with 32P as described for the nuclease-resistance assay and then electrophoresed on a non-denaturing 20% polyacrylamide gel to check labeling. Whole-cell extracts from A549, DU145, MCF7 and Hs27 cells were prepared using a modification of a previously published method (17) as follows: cells were grown until confluent in 75 cm2 flasks. The cells were washed, then scraped with 100 µl HEDG buffer [20 mM HEPES-NaOH, pH 7.8, 0.5 mM EDTA, 0.5 mM dithiothreitol (DTT)] containing 10% glycerol, 1 × protease inhibitor cocktail (Sigma, St Louis), and 0.42 M NaCl. The solution was harvested and subjected to three freeze–thaw cycles (liquid nitrogen versus room temperature). Samples were centrifuged at 16 000g at 4°C for 45 min to pellet cell debris. An equal volume of HEDG with glycerol and protease inhibitor but without NaCl was added to reach a final concentration of 0.21 M NaCl. An aliquot (100 000 c.p.m.) of radiolabeled oligomer was combined with 5.0 μg of cell extract from A549, DU145 and MCF7 cells, or with 3.2 μg from Hs27 cells, and 5X protein binding buffer [1X is 20 mM Tris–HCl, pH 7.4, 140 mM Kl, 2.5 mM MgCl2, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 8% (v/v) glycerol]. Samples were incubated at 37°C for 15 min and run on a non-denaturing 5% polyacrylamide gel. Gels were fixed in a solution of 10% (v/v) methanol + 10% (v/v) glacial acetic acid in water for 10 min at room temperature, then exposed to a storage phosphor screen for 2 h at room temperature before scanning. Gels were also exposed to X-ray film overnight at −80°C. RESULTS AND DISCUSSION GT libraries of all types and the GC 26mer library have selective antiproliferative activity against cancer cells.

The antiproliferative activity of 11 oligomer libraries and three monobasic oligomers at various concentrations against four cell lines was determined using the MTT assay. The results shows the percentage reduction in cell number at 6-µM oligonucleotide strand concentration. This format was selected for clarity of presentation and 6 µM was chosen because it best illustrated the relative activities of the various libraries.

ANTIPROLIFERATIVE ACTIVITY OF LIBRARIES AND OLIGOMERS (A) Tabulation of percent decrease in cell number at 6 µM (strand concentration), calculated taking untreated wells to be 100%. Dark-gray shading indicates >50% decrease; light-gray shading indicates between 30% and 50% decrease. (B) Growth curves showing the effects of all libraries, non-G-rich and G-rich, on non-cancerous fibroblasts (Hs27). Relative cell numbers at each concentration are compared to no treatment. (C) Growth curves showing the effects of GT libraries of different lengths on A549 lung cancer cells. The N library is shown for comparison. (D) Growth curves showing the effects of various GT libraries on A549 lung cancer cells. The N library is shown for comparison. (E) Growth curves showing the effects of the GC 26-mer library on four cell lines.

Some libraries containing G+T or G+C were found to strongly inhibit cancer cell proliferation at low micromolar concentrations, whereas non-G-rich libraries, including the standard N library, had little effect on the cancer cells. All libraries tested had minimal antiproliferative activity against the non-cancerous Hs27 cells. These 250

RIBOGRAMA PROJECT novel data provide surprising evidence that some oligomer libraries possess intrinsic antiproliferative activity, even before isolation of individual aptamer sequences. The GT 18-mer, 26-mer and 34-mer libraries had the highest level of antiproliferative activity against the three cancer cell lines. The GT 10-mer, GT 0.33 26-mer and GT 0.67 26-mer libraries had similar specificity, but were less effective at inhibiting the cancer cells. The GC 26-mer library had potent antiproliferative activity against A549 and DU145 cells, and had a weaker effect on MCF7 cells. In contrast to the other G-rich libraries, the GA 26-mer library had very little antiproliferative activity against any cell line. Based on the shape of the dose-dependence curves, it appears that active libraries have a cytostatic rather than cytotoxic effect. This was also observed previously for individual antiproliferative G-rich oligonucleotides (16).

From these data, we designated all of the GT libraries and the GC 26-mer library as ‘active’ against cancer cells. In the remaining experiments, we compared these active libraries against the remaining ‘inactive’ libraries in order to identify characteristics that were associated with the unexpected antiproliferative activity.

To confirm the cancer-selectivity of the active libraries, we also examined all libraries and oligomers in an additional non-tumorigenic cell line, MCF10A, which is derived from benign breast epithelial cells. The results indicated that although the MCF10A cells were slightly more sensitive to antiproliferative effects than the Hs27 skin fibroblasts, they were markedly less sensitive than the cancer cell lines, including MCF7 breast cancer cells.

LIBRARIES WITH ANTIPROLIFERATIVE ACTIVITY SHARE FEATURES OF CD SPECTRA CD of DNA arises from the helical arrangement of the bases and therefore conformational characteristics (e.g.

A-, B- or Z-forms of duplexes, and C- or G-quadruplexes) can often be inferred from CD spectra, as described in an excellent recent review article (18). In our experiments, we observed that the CD spectra of active libraries typically displayed a negative peak at ∼240 nm, a strong positive peak at 260–265 nm and an additional maximum or shoulder (depending on the cation present) in the region 285–290 nm. In addition, the spectra of the active libraries changed significantly between different salt solutions, with the largest peaks seen in the 100 mM potassium chloride solution. While CD data alone cannot be used to absolutely assign structure, it is widely accepted that positive peaks close to 260 nm or 290 nm are associated with Gquadruplex formation (18,19). Similarly, the conformational polymorphism suggested by the differences in CD spectra in the presence of various cations is typical of G-quadruplexes. Obviously, the shape of the spectrum for an oligonucleotide library is derived from the combined CD spectra from millions of sequences. Thus, the characteristic spectrum of the active GT libraries presumably represents contributions from molecules with various arrangements of G-quartets (maxima at 260 and 290 nm) and non-quadruplex single-stranded molecules (maximum at 275–280 nm). Somewhat surprisingly, the spectrum of the GC library was quite similar to the GT libraries and shared features suggestive of G-quadruplex formation, although the presence of duplexes or hairpins in the GC-library cannot be excluded.

CD STUDIES Samples were prepared at a concentration of 5 µM in three different solutions: water (blue), 100 mM NaCl (red) and 100 mM KCl (green). (A) CD spectra of various active libraries showing a similar profile (a positive peak at 260 nm and a second positive peak or shoulder at 290 nm), which is characteristic of G-quadruplex formation. (B) CD spectra of inactive libraries showing a similar profile (positive peak in 270–280-nm region), characteristic of non-G-quadruplex oligonucleotides. (C) Libraries with anomalous CD. The inactive GA 26-mer library CD is different from both active and inactive profiles. The GT 0.33 26-mer library has some antiproliferative activity, but its CD spectrum is similar to that of the inactive libraries. Results are shown as molar CD (Δε), referring to per mole of nucleotide.

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Most of the inactive libraries showed a negative peak at ∼250 nm as well as a single positive peak at ∼275 nm. This is typical of both ordered single-stranded and double-stranded DNA (18). The spectra of the inactive libraries changed only minimally with different salt solutions, except for the CT library and (dA) 26, which showed slightly different spectral patterns in water compared to the salt-containing solutions. Interestingly, the (dC) 26 oligomer displayed a very large ellipticity and the peaks were slightly phase-shifted from the remaining inactive libraries, with a negative peak at 265 nm and positive at 285 nm. This subtle shift in CD spectra may be due to the formation of secondary structures called i-motifs that have been reported in cytosine-rich molecules (20,21), albeit usually only under slightly acidic conditions. Our theory was supported by additional experiments in buffered solutions, which showed that the amplitude and shape of the CD spectrum for (dC)26 was strongly pH-dependent and was typical of the i-motif (18,21) at pH 5, 6 or 7, but there was a transition to the standard single-stranded DNA spectrum at pH 8. The two exceptions to the general trends were the GT 0.33 26-mer library and the GA 26-mer library; although the GT 0.33 26-mer library is included in the active libraries, its spectrum resembled the inactive libraries, consistent with the expected much smaller proportion of quadruplex-forming sequences and its lower level of antiproliferative activity. The spectrum of the inactive GA 26-mer library varied considerably between water, NaCl and KCl, but was distinct from any of the other libraries examined, being close to zero at 240 nm, with a positive peak at 260 nm and a negative peak at 285 nm. This has been reported to be typical of G + A containing oligonucleotides and associated with stacked guanosines, although it is not clear if this reflects the presence of quadruplexes or other ordered structures, such as alternatively base-paired duplexes (18).

Libraries with antiproliferative activity are significantly more stable in serum than inactive libraries To assess nuclease resistance, oligonucleotides and libraries were radiolabeled at the 5′-end and incubated in complete medium (which contains serum-derived exonucleases) for various times. The primary purpose of this experiment was to determine nuclease stability under the exact conditions used in the MTT assays. This was considered essential, because if an oligonucleotide is degraded in the cell culture medium, it is unlikely to be active whatever other properties it has (unless the active component is derived from degradation of the oligonucleotide into nucleotides). The results showed that libraries that did not contain any G bases, as well as all of the monobasic oligomers tested, were highly susceptible to degradation in serum-containing medium, with most being fully degraded within 90 min. Libraries containing guanines in any percentage had at least some molecules still intact at 72 h, regardless of antiproliferative activity.

NUCLEASE-RESISTANCE IN SERUM-CONTAINING MEDIUM (A) Autoradiographs showing electophoretic migration of radiolabeled oligomers or libraries following incubation in complete cell culture medium, which contains serum exonucleases. (B) Mean stability of active versus inactive libraries. The difference in band density between active and inactive libraries was statistically significant at 24 and 72 h (*P < 0.05 using Student’s two-tailed t-test). Although all active libraries displayed stability in the presence of serum nucleases, the degree of stability did not correlate with antiproliferative activity. For example, the moderately active GT 10-mer library was markedly less stable than the inactive N 26-mer library. However, although band density did not correspond to antiproliferative activity for the individual libraries, the mean band density of the active libraries at 24 and 72 h was significantly greater (P = 0.026 using Students’ two-tailed t-test) than the inactive libraries. Active libraries had increased cellular uptake compared to inactive libraries as shown by flow cytometry.

For this assay, four representative 26-mer libraries (the active GT and GC libraries, and the inactive N and GA libraries) were synthesized with a fluorophore at the 3′-end and used to assess cellular uptake by flow cytometry. Cells were washed and harvested with trypsin prior to analysis to remove any surface-bound oligonucleotides. The relative level of uptake in a given cell line can be estimated by determining the percent 252

RIBOGRAMA PROJECT of cells that are ‘positive’ for the fluorophore (i.e. have a greater fluorescent intensity than the background signal, which arises from autofluorescence of the cells) after excluding non-viable cells. This type of analysis is especially useful for comparing the uptake of various libraries and oligonucleotides in a particular cell line (22), although its use for comparing uptake between different cell lines is somewhat complicated by differing levels of baseline autofluorescence between cell lines.

We observed that the active libraries, GT and GC, consistently yielded a higher proportion of fluorescent cells compared to inactive libraries in all cell lines. In general, the inactive N library had the lowest uptake of any library examined, and the inactive GA library had a similar or slightly higher uptake than the N library, but substantially less than the active libraries. For the GC 26-mer library, whereas its uptake in Hs27, DU145 and A549 cells was close to that of the GT library (albeit a different distribution), its uptake in MCF7 cells was considerably less than the GT library. This result is noteworthy because the GC library also has reduced antiproliferative activity in this particular cell line. Thus, these data suggest a correlation between relative uptake and antiproliferative activity, although with the caveat that, as mentioned above, this technique may not be optimal for comparing uptake between different cell lines. We noted the non-uniform shape of the curve for the GT library compared to other libraries, which may indicate a non-uniform uptake of the GT library (some cells take up much more oligomer than others), but the reason for this is unclear at present.

CELLULAR UPTAKE OF LIBRARIES AND OLIGOMERS Fluorescently labeled oligonucleotides (5 µM strand concentration) were incubated with cells for 1 h. Cells were harvested and stripped of surface-bound DNA using trypsin, then analyzed by flow cytometry to determine uptake. Uptake was quantified by determining the percentage of cells included in the black gate (positive for FAM after controlling for autofluorescence). (A) Uptake in Hs27 non-malignant fibroblasts. (B) Uptake in A549 cancer cells. (C) Uptake in DU145 cancer cells. (D) Uptake in MCF7 cancer cells. (E) Table of percent of FAM-positive cells for each sample.

Active libraries and inactive libraries had quantitative and qualitative differences in protein binding The protein binding patterns of 5′-radiolabeled libraries or oligonucleotides following incubation with cell extracts were examined by electrophoretic mobility shift assay (EMSA). To start, we first analyzed the libraries and oligomers in the absence of proteins. The results confirmed the labeling and integrity of each sample and showed a quite marked dependence of electrophoretic migration on base composition, as expected. A very general observation from the EMSA experiments was that inactive libraries and oligomers displayed fewer and less intense shifted bands, when compared to the active libraries across all samples. Appearance of the band marked ‘B1’ was associated for the most part with nuclease-resistance libraries, but not nuclease-susceptible libraries or oligomers, suggesting it may be a general DNA-binding protein. However, this was usually most intense for the most active libraries and was present at much higher levels in the cancer cell lines compared to the non-cancer cells, suggesting it may contribute to the antiproliferative effects. The series of faster migrating bands indicated by ‘B2’ were much less intense than B1, but are of interest because they were present predominantly for the most active libraries (GT26 and GT34). However, these bands were observed for Hs27 cell extracts, as well as for cancer cells. The band marked ‘B3’ is particularly interesting because it is specific for the GC-library, which has strong antiproliferative activity, and it is present at high levels in all of the cancer cells, but is undetectable in the non-malignant cells. The fastest migrating band we have highlighted, ‘B4’, is present only in the C26 sample. Although this sequence has no antiproliferative activity, this band is interesting because oligo–dC sequences may form unusual structures, involving the i-motif, and very few proteins that bind specifically to such structures have been previously reported (20). We plan to follow up these studies in the future by attempting to identify proteins of interest using mass spectrometry-based techniques.

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RIBOGRAMA PROJECT PROTEIN BINDING OF LIBRARIES AND OLIGOMERS Radiolabeled oligonucleotides were incubated with whole-cell extracts and analyzed by native gel EMSAs. Oligonucleotides were with: (A) no protein extracts (oligomers alone); (B) A549 extracts; (C) DU145 extracts; (D) MCF7 extracts; (E) Hs27 extracts; or (F) representative libraries were directly compared for binding to extracts (5 μg) from the four cell types. The arrows indicate DNA–protein complexes of particular interest (see text for discussion), and ‘O’ indicates the origin of the gel.

Interpretation of the EMSA data in the presence of protein has the potential to be somewhat complicated because the presence of shifted bands may depend not only on protein binding activity, but on nucleaseresistance as well. The relative nuclease-susceptibility under these conditions was expected to reflect the results from our serum stability assay (Figure 5), but may not be exactly the same because cell extracts contain endonucleases, as well as exonucleases. To further investigate these issues, we performed nucleaseresistance assays in the presence of the DU145 cellular extracts that were used in the EMSA experiments. We examined stability after incubation for a short time (15 min), which mimics the EMSA conditions, and found no significant nuclease degradation. These results verify that the absence of a shifted band in the EMSA is due to the complex not being formed, rather than the oligonucleotides being degraded by nucleases. After longer incubation (18 h), which we anticipate will reflect the persistence of oligonucleotides inside cells, we observed relative stabilities similar to those reported above for long-term stability in cell culture medium. This confirms our conclusions regarding the relationship between nuclease resistance, protein binding and antiproliferative activity (i.e. that, while active libraries are invariably stable and display strong protein binding, not all libraries that are stable and display strong protein binding are active). Furthermore, the data also suggest that protein binding may be responsible for nuclease resistance and not vice versa.

CONCLUSIONS In this study, we have examined the biological properties and CD spectra of oligonucleotide libraries. The purpose of this exercise was primarily to test our hypothesis that G-rich libraries would be enriched in quadruplexes, which might make them useful starting points for the development of new therapeutic aptamers using SELEX or similar technologies. The results of our analyses seem to confirm that G-rich oligodeoxynucleotide libraries do indeed contain large numbers of quadruplex-forming sequences, and they are more nuclease-resistant and efficiently internalized by cells compared to the standard ‘N library’ currently used in SELEX. Experiments are now underway to determine if G-rich libraries can be used for SELEX and whether they will allow faster evolution of aptamers for in vivo use.

While many of the observed properties of G-rich libraries were anticipated, the strong and cancer-selective growth inhibitory effects of some GT and GC libraries were unforeseen. When analyzing a library of molecules, it is important to remember that the results are the averages of the effects of all the different molecules within the library. Given our previous work, showing that several different quadruplex-forming oligonucleotides have cancer-selective antiproliferative activity (19,23), we expected that G-rich libraries might contain a small proportion of active molecules. However, while we anticipated that a G-rich library might contain some very potent molecules, we presumed that the vast majority of the estimated 67 000 000 sequences in such a library would be ineffective, such that the library as a whole would display at best a modest growth inhibitory effect against cancer cells. Thus, the finding that certain GT and GC libraries have very potent and tumor-selective antiproliferative effects against a variety of cancer cell types was somewhat surprising. Remarkably, the antiproliferative activity of these libraries was comparable to some individual G-rich oligodeoxynucleotides that are currently in pre-clinical development or clinical trials (11).

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RIBOGRAMA PROJECT Although a full understanding of the antiproliferative properties of active libraries will require further efforts, the work that we have described provides some clues to the mechanism of action for these effects. The restriction of the activity to only certain GT and GC libraries indicates that it cannot be a non-specific effect of phosphodiester oligonucleotides or simply due to the presence of deoxyguanosine nucleotides (either in the context of an oligonucleotide or as nucleobases resulting from oligomer degradation), because some dGcontaining libraries, including the GA and N libraries, had no activity. In addition, although active libraries are generally more stable than inactive libraries, nuclease-resistance alone cannot explain the antiproliferative effects because some of the inactive libraries displayed comparable stability.

In view of our observations, we now propose that antiproliferative activity requires all of the following: nuclease-resistance, efficient cellular uptake and binding to a specific protein (or proteins). It is not yet clear if quadruplex formation, per se, is necessary for activity, or whether it is simply that quadruplex-forming oligonucleotides generally have the aforementioned properties. One possible candidate for the complex in the EMSAs that appears to be most relevant for activity is nucleolin. This protein is highly expressed in cancer cells and is a general DNA binding protein, but binds particularly strongly to a variety of quadruplex-forming sequences (24â&#x20AC;&#x201C;27). It is also the molecular target of an existing G/T oligonucleotide aptamer (11). However, further studies will be required to verify this possibility and to reconcile why some libraries bind to this protein (if it is indeed band B1), yet do not have enhanced cellular uptake or antiproliferative activity. Interestingly, the GC library appears to have a different cancer cell selectivity compared to the GT libraries and forms a specific protein complex, suggesting that its mechanism of action and molecular target may be distinct from the GT oligonucleotides.

In summary, the potent intrinsic and selective antiproliferative activity of the GT and GC libraries is an unexpected and wholly novel finding. Our results indicate that cancer-selective antiproliferative activity is a common characteristic of G-rich DNA oligonucleotides, which may explain why numerous different G-rich oligonucleotides have been reported as potential anticancer agents (11). Although many different mechanisms of action have been proposed for these, our results suggest there may be common mechanisms (related to nuclease resistance, protein binding and cellular uptake) that contribute to the activity of G-rich oligonucleotides. Further research is warranted to elucidate the mechanism of action for these cancerselective effects, as this may lead to insights into basic cancer cell biology, as well as to the identification of novel molecular targets and new oligomer-based therapies for cancer.

FUNDING The National Institutes of Health (R01CA122383 to P.B.); the Department of Defense Prostate Cancer Research Program (fellowship PC073700 to E.C.); the Kentucky Lung Cancer Research Program (to P.B.); the Institute for Molecular Diversity and Drug Design (fellowship to E.C.).

Funding for open access charge: Department of Defense [Prostate Cancer Research Program training grant (to E.C. and P.B.)].

Conflict of interest statement. P.B. has a financial interest in Antisoma, PLC (London, England) as a shareholder, consultant, and recipient of research support. P.B. has a financial interest in Transmed Oncology, Inc. as a shareholder, consultant, and recipient of research support.

This is an Open Access article distributed under the terms of the Creative Commons Attribution NonCommercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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